Corynebacteria

II MBBS

Dr Ravi Shankar Gupta

Microbiology
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
 Historical overview I.
Corynebacterium diphtheriae
Bretonneau 1826 Behring and Kitasato
Clinical characterisation 1890-1892
of diphtheria – diphtherite - Discovering the diphtheria
antitoxin
Klebs 1883 - Antitoxic immunity (therapy and
Detecting the bacterium prevention)

Loeffler 1884 Roux 1894

Isolating the bacterium Treatment with antitoxin

Roux and Yersin 1888

Discovering the diphtheria toxin
Introduction – C. diphtheriae

 Diphtheros – leather (tough, leathery

pseudomembrane)

 Also known as Klebs–Loeffler bacillus

 Causes Diphtheria
Important features of C. diphtheriae
 Slender Gram positive bacilli

 Pleomorphic, non motile, non sporing

 Chinese letter or Cuneiform arrangement

 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 Loeffler’s Methylene
blue
 Special stains: Albert’s, Neisser’s &
Ponder’s

 Grows aerobically at 37°C

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
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
Diphtheria Toxin
 Toxigenicity can be induced by Lysogenic or phage
conversion – corynephages (tox+ phage) or beta
phages

 Can be toxoided by -
1. Prolonged storage
2. Incubation at 37°C for 4 - 6 weeks
3. Treatment with 0.2 – 0.4 % formalin or
4. Acid pH.

 Stain used for toxin production – ‘Park Williams 8’ strain

 Antibodies to fragment B - protective

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
Diphtheria
 Site of infection
1. Faucial (palatine tonsil) – commonest type
2. Laryngeal
3. Nasal
4. Conjunctival
5. Genital – vulval, vaginal
6. Cutaneous – caused by non toxigenic strains
Pathogenesis & Clinical Manifestations
 Human Disease
1. Usually begins in respiratory tract
2. Virulent diphtheria bacilli lodge in throat of
susceptible individual
3. Multiply in superficial layers of mucous
membrane
4. Elaborate toxin which causes necrosis of
neighboring tissue cells
5. 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)
Dr Ekta, Microbiology
Diphtheria - Clinical Classification
 Based on the severity of clinical presentation:
1. Malignant or hypertoxic – severe toxemia with
marked adenitis

2. Septic – ulceration, cellulitis, & gangrene around the

pseudomembrane

3. Hemorrhagic – bleeding from the edge of

membrane, epistaxis, conjunctival hemorrahge,
purpura & generalized bleeding tendency.
 Complications of diphtheria
 Mechanical complications are due to the
pseudomembrane, while the systemic effects
are due to the toxin.
1. Asphyxia – due to obstruction of respiratory
passage
2. Acute circulatory failure
3. Postdiphtheritic paralysis – occurs in 3rd or 4th
week of disease, palatine & ciliary, spontaneous
recovery
4. Sepsis – pneumonia & otitis media

14.12.08 Dr Ekta, Microbiology

14.12.08 Dr Ekta, Microbiology
 Laboratory Diagnosis
 Specimen – swab from the
lesions

1. Microscopy
 Gram stain: Gram +ve bacilli,
chinese letter pattern
 Immunofluorescence
 Albert’s stain for
metachromatic granules

14.12.08 Dr Ekta, Microbiology

Laboratory Diagnosis
2. Culture – isolation of bacilli requires media
enriched with blood, serum or egg
a. Blood agar
b. Loeffler’s serum slope – rapid growth, 6 to 8 hrs
c. Tellurite blood agar – tellurite is reduced to
tellurium, gives gray or black color to the colonies
d. Hoyle’s media modifications of TBA
e. McLeod’s media

14.12.08 Dr Ekta, Microbiology

 Growth of diphtheria bacilli

Blood agar

Tellurite blood agar

Loeffler’s serum slope

14.12.08 Dr Ekta, Microbiology
 Biotypes of Diphtheria bacilli
 Based on colony morphology on the tellurite medium &
other properties, McLeod classified diphtheria bacilli
into three types:
Features 1. Gravis 2. Intermedius 3. Mitis

Case fatality rate High High Low

Complications Paralytic, Hemorrhagic Obstructive

hemorrhagic
Predominance In epidemic areas Epidemic areas Endemic areas

Spread Rapid Rapidly than mitis Less rapid

Colony on TBA ‘Daisy head” ‘Frog’s egg ‘Poached egg’

colony colony colony
Hemolysis Variable Nonhemolytic Usually
hemolytic
Laboratory Diagnosis

3. Biochemical reactions
a. Hiss's serum water - ferments sugar with acid
formation but not Gas
ferments: glucose, galactose, maltose and dextrin

b. Resistant to light, freezing

c. Sterilization: sensitive to heat (destroyed in 10mins

at 58°C or 1min in 100°C), chemical disinfectants
Laboratory Diagnosis
4. Virulence tests - Test for toxigenicity
A. Invivo tests – animal inoculation (guinea
pigs)
a. Subcutaneous test
b. Intracutaneous test

B. Invitro tests
a. Elek’s gel precipitation test
b. Tissue culture test
 Laboratory Diagnosis
Virulence tests - Invivo tests
 Bacterial growth from Loeffler’s serum slope is emulsified in 2-4 ml
broth.
 Two guinea pigs (GP A and GP B)

I. 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 four hrs after the skin test
 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
 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

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
Prophylaxis
1) Active Immunization (Vaccination)

i. Formol toxoid (fluid toxoid)

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

ii. Adsorbed toxoid (more immunogenic than fluid

toxoid)
 purified toxoid adsorbed onto insoluble aluminium
phosphate or aluminium hydroxide
 given IM (DTP)
Prophylaxis
 Adsorbed Toxoid
a. DPT - triple vaccine given to children; contains diphtheria toxoid,
Tetanus toxoid and pertussis vaccine

b. DaT - contains absorbed tetanus and ten-fold smaller dose of

diphtheria toxoid. (smaller dose used to diminish likelihood of
adverse reactions)

 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

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
CONTROL
1. isolate patients
2. treat with antibiotics actively
3. complete vaccination schedule should be
used with booster every 5 years
Other Corynebacteria
 C. ulcerans – diphtheria like lesions in guinea pigs &
cows, may get transmitted to humans by cow’s milk

 Diphtheroids –
 Normal commensals of nose, throat, nasopharynx, skin, urinary
tract & conjunctiva
 Stain uniformly
 Few or no metachromatic granules
 Arranged in parallel rows
 Nontoxigenic