Corynebacteria

II MBBS Dr Ekta Chourasia Microbiology

Gram positive rods

Non spore-forming 1. AEROBIC

Spore-forming 1. AEROBIC

Corynebacteria

Genus: Bacillus

C. diphtheriae diphtheroids C. jeikeium

2.

B. anthracis B. cereus B. subtilis

Listeria monocytogenes Erysipelothrix rusiopathiae

2.

ANAEROBIC

Genus: Clostridium

ANAEROBIC

Lactobacillus spp.

C. tetani C. botulinum C. difficile C. perfringens

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Corynebacteria - Overview

Gram positive, non motile bacilli with irregularly stained segments

Frequently show club shaped swellings corynebacteria (coryne = club) C. diphtheriae : most important member of this genus, causes diphtheria

Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva.

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Historical overview I. Corynebacterium diphtheriae

Bretonneau 1826 Clinical characterisation of diphtheria diphtherite
Klebs 1883 Detecting the bacterium Loeffler 1884 Isolating the bacterium

Behring and Kitasato 1890-1892 - Discovering the diphtheria antitoxin - Antitoxic immunity (therapy and prevention)
Roux 1894 Treatment with antitoxin

Roux and Yersin 1888 Discovering the diphtheria toxin

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Historical overview I. Corynebacterium diphtheriae

Emil von Behring 1901 Nobel prize
Behring 1913 Active immunisation I. with toxin-antitoxin mix

Ramon 1923 Active immunisation II. Anatoxin = toxoid

Freeman 1951 PHAGE (lysogenia, toxin production)

Schick 1913 Skin test

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Introduction C. diphtheriae

Diphtheros leather (tough, leathery pseudomembrane) Also known as KlebsLoeffler bacillus

Causes Diphtheria

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Important features of C. diphtheriae

Slender Gram positive bacilli Pleomorphic, non motile, non sporing Chinese letter or Cuneiform arrangement Stains irregularly, tends to get easily decolorised May show clubbing at one or both ends Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules Metachromatic Granules:

made up of polymetaphosphate Bluish purple color with Loefflers Methylene blue Special stains: Alberts, Neissers & Ponders

Grows aerobically at 37C

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Virulence factor

Exotoxin Diphtheria toxin:

Protein

in nature very powerful toxin Responsible for all pathogenic effects of the bacilli Produced by all the virulent strains Two fragments A & B

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Diphtheria toxin Mechanism of action

DT - Acts by inhibition of protein synthesis

Fragment A inhibits polypeptide chain elongation by inactivating the Elongation factor EF 2 in the presence of NAD
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Diphtheria Toxin

Toxigenicity can be induced by Lysogenic or phage conversion corynephages (tox+ phage) or beta phages Can be toxoided by 1. 2. 3. 4.

Prolonged storage Incubation at 37C for 4 - 6 weeks Treatment with 0.2 0.4 % formalin or Acid pH.

Stain used for toxin production Park Williams 8 strain Antibodies to fragment B - protective
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Epidemiology

Habitat nose, throat, nasopharynx & skin of carriers and patients Spread by respiratory droplets, usually by convalescent or asymptomatic carriers Nasal carriers harbour the bacilli for longer time than pharyngeal carriers Local infection of throat - toxemia Incubation period of diphtheria 3 to 4 days In tropics, cutaneous infection is more common than respiratory infection

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Diphtheria

Site of infection
1. 2. 3. 4. 5. 6. 7.

Faucial (palatine tonsil) commonest type Laryngeal Nasal Otitic Conjunctival Genital vulval, vaginal, prepucial Cutaneous usually a secondary infection on preexisting lesion, caused by non toxigenic strains

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Pathogenesis & Clinical Manifestations

1. 2. 3. 4. 5.

Human Disease
Usually begins in respiratory tract Virulent diphtheria bacilli lodge in throat of susceptible individual Multiply in superficial layers of mucous membrane Elaborate toxin which causes necrosis of neighboring tissue cells Inflammatory response eventually results in pseudomembrane (fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria)

Usually appears first on tonsils or posterior pharynx and spreads upward or down In laryngeal diphtheria, mechanical obstruction may cause suffocation Regional lymphnodes in neck often enlarged (bull neck)

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Diphtheria - Clinical Classification

Based on the severity of clinical presentation:

1.

Malignant or hypertoxic severe toxemia with marked adenitis Septic ulceration, cellulitis, & gangrene around the pseudomembrane
Hemorrhagic bleeding from the edge of membrane, epistaxis, conjunctival hemorrahge, purpura & generalized bleeding tendency.

2.

3.

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Complications of diphtheria

Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin.
1. 2. 3.

4.

Asphyxia due to obstruction of respiratory passage Acute circulatory failure Postdiphtheritic paralysis occurs in 3rd or 4th week of disease, palatine & ciliary, spontaneous recovery Sepsis pneumonia & otitis media

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Laboratory Diagnosis

Specimen swab from the lesions Microscopy

1.

Gram stain: Gram +ve bacilli, chinese letter pattern Immunofluorescence Alberts stain for metachromatic granules

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Laboratory Diagnosis
2.

Culture isolation of bacilli requires media enriched with blood, serum or egg
a. b.

c.
d. e.

Blood agar Loefflers serum slope rapid growth, 6 to 8 hrs Tellurite blood agar tellurite is reduced to tellurium, gives gray or black color to the colonies Hoyles media modifications of TBA McLeods media

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Growth of diphtheria bacilli

Blood agar

Tellurite blood agar

Loefflers serum slope

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Biotypes of Diphtheria bacilli

Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types:
Features Case fatality rate Complications 1. Gravis High Paralytic, hemorrhagic 2. Intermedius High Hemorrhagic 3. Mitis Low Obstructive

Predominance
Spread

In epidemic areas Epidemic areas

Rapid

Endemic areas

Rapidly than mitis Less rapid

Colony on TBA
Hemolysis
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Daisy head colony

Variable

Frogs egg colony

Nonhemolytic

Poached egg colony

Usually hemolytic

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Laboratory Diagnosis
3.

Biochemical reactions
a.

Hiss's serum water - ferments sugar with acid formation but not Gas ferments: glucose, galactose, maltose and dextrin Resistant to light, desiccation and freezing

b. c.

Sterilization: sensitive to heat (destroyed in 10mins at 58C or 1min in 100C), chemical disinfectants

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Laboratory Diagnosis
4.

Virulence tests - Test for toxigenicity

A.

Invivo tests animal inoculation (guinea pigs)

a. b.

Subcutaneous test Intracutaneous test

B.

Invitro tests
a. b.

Eleks gel precipitation test Tissue culture test

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Laboratory Diagnosis
Virulence tests - Invivo tests
I.

Bacterial growth from Loefflers serum slope is emulsified in 2-4 ml broth. Two guinea pigs (GP A and GP B) Subcutaneous test 0.1 ml of emulsion is injected SC into each guinea pig
GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B - Doesn't have antitoxin

II.

Intracutaneous test - 0.1 ml of emulsion is injected IC into each guinea pig

GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B 50 units of antitoxin IP four hrs after the skin test

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Laboratory Diagnosis
Virulence tests - Invitro tests
I.

Elek's gel precipitation test

filter paper saturated with antitoxin (1000units/ ml) is placed on agar plate with 20% horse serum bacterial culture streaked at right angles to filter paper

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Laboratory Diagnosis
Virulence tests - Invitro tests
II.

Tissue culture test - incorporation of bacteria into agar overlay of

eukaryotic cell culture monolayers. Result: toxin diffuses into cells and kills them

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Treatment

specific treatment must not be delayed if clinical picture suggests of diphtheria

rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin) early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV
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Prophylaxis
1)

Active Immunization (Vaccination)

i.

Formol toxoid (fluid toxoid)

incubation of toxin with 0.3% formalin at pH 7.4 - 7.6 at 37C for 3 to 4 weeks fluid toxoid is purified and standardized in flocculating units (Lf doses)

ii.

Adsorbed toxoid (more immunogenic than fluid toxoid)

purified toxoid adsorbed onto insoluble aluminium phosphate or aluminium hydroxide given IM (DTP or TD)

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Prophylaxis

Adsorbed Toxoid
a.

DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine DaT - contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid. (smaller dose used to diminish likelihood of adverse reactions)

b.

Schedule i) Primary immunization - infants and children

- 3 doses, 4-6 weeks interval - 4th dose after a year - booster at school entry

ii) Booster immunization - adults -Td toxoids used (travelling adults may need more)

SHICK test - to test susceptibility to vaccine, not done now-a-days

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Prophylaxis
2.

Passive immunization
ADS (Antidiphtheritic serum, antitoxin) made from horse serum - 500 to1000 units subcutaneously

3.

Combined immunization
First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation

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CONTROL
1.
2. 3.

isolate patients treat with antibiotics actively complete vaccination schedule should be used with booster every 5 years

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Other Corynebacteria

C. ulcerans diphtheria like lesions in guinea pigs & cows, may get transmitted to humans by cows milk
Diphtheroids

Normal commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva Stain uniformly Few or no metachromatic granules Arranged in parallel rows (palisades) Nontoxigenic

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