Corynebacterium diphtheriae

Prof. Mochammad Hatta, MD, Ph.D, Clin Micro (Cons)

Dept Microbiology, Molecular Biology and Immunology
Laboratory, Fac.Medicine, Hasanuddin University,
Makasssar
Corynebacterium
Family : Corynebacteriaceae
Genus : Corynebacterium
Classification
Corynebacterium diphtheriae and diphtheroids
(look like C. diphtheriae) are Gram- positive,
club shaped rods.
Some are saprophytic
Some produce disease in animals.
C. diphtheriae is the most important pathogen in the group.
Morphology and cultural characteristics
Small G+B; arrangement=palisade or Chinese
letters
Growth on B.A raised, translucent, gray colonies
Diphtheroids Gram stain
 Biological Features

Aerobic, Gram+, Noncapsulated, rods

Gray-black colonies on tellurite medium
Metachromatic granules

Chinese-letter morphology in Gram stain

Arrangement of C. diphtheria
Corynebacterium

Loefflers agar slant contains serum and

egg that enhance the formation of
metachromatic granules (polymerized
polyphosphoric acid) in C. diphtheriae.
Also called Babes-Ernst granules.
They are visualized by staining with
methylene blue.
Methylene blue stain from
Loefflers slant
Corynebacterium
A medium containing tellurite should be used to
select for Corynebacterium and other
G+ organisms -it inhibits G organisms. Two kinds
are used:
Cystine tellurite has a longer shelf life
Tinsdale helps to differentiate amongst the
Corynebacterium.
Colonies on either appear black or gray due to tellurite
reduction.
S. aureus and Listeria also grow as black colonies.
On Tinsdale C. diphtheriae, ulcerans, and
pseudotuberculosis form brown halos around the colonies
due to formation of ferric sulfide.
Corynebacterium
3 morphological types of C. diphtheriae are found
on tellurite containing media:
Mitis black colonies with a gray periphery
Gravis large, gray colonies
Intermedius small, dull gray to black.
All produce an immunologically identical toxin.
Incubation -35-370 C for 24 hours.
They prefer a pH of 7.8-8.0 for good growth.
They require access to oxygen (poor AnO2 growth).
Biochemistry
Catalase +
Corynebacterium
Nonmotile
C. ulcerans is urease +, C. diphtheriae is -, and C.
pseudotuberculosis is usually +
Virulence factors C. diphtheriae
For C. diphtherias to cause diphtheria an exotoxin
must be produced.
Is a heat-labile polypeptide produced during lysogeny of a
phage that carries the "tox gene.
Alkaline pH of 7.8- 8.0, aerobic conditions, and a low
environmental iron level are essential for toxin production
(occurs late in the growth of the organism).
The toxin inhibits protein synthesis by ADP-ribosylating
elongation factor 2.
What other organism produces a similar toxin?
Corynebacterium
Trypsin cleaves the toxin into 2 fragments, A and B, that
are linked together by a disulfide bridge.
Fragment B is required for toxin binding to tissue cells
and fragment A contains the toxic activity.
One molecule of toxin can inhibit 90% of the protein
synthesis in a cell.
Systemic effects include heart failure, paralysis and
adrenal hypofunction leading to an Addisons like disease.
C. ulcerans and C. pseudotuberculosis sometimes
make a diphtheria-like toxin.
Immunity
Immunization of animals with altered toxin,
producing antitoxin, was first done in
1890, 1st used in humans in 1891
Toxin-antitoxin introduced by Theobald
Smith in 1909, used little
Toxoid introduced in 1923, now widely
used
C. diphtheria toxin

Toxin enters through

receptor mediated
endocytosis
Acidification of
endocytic vesicle
allows A to
dissociate from B
A enters cycoplasm
 Diphtheria Toxin (DT)
Cleaved to yield A/B fragment, joined by bond
- A (catalytic domain)
- B (transmembrane and receptor binding domains)
Receptor: heparin-binding epidermal growth factor - rich on cardiac cells and nerve
cells
Toxin diffuses throughout body via blood
- Cardiac, neurologic complications
- Heart/respiratory damage, paralysis
C. diphtheria toxin
Corynebacterium
To prove that an isolate can cause
diphtheria, one must
demonstrate toxin production.
This is most often done on an Elek plate:
The organism is streaked on a plate containing low
iron.
A filter strip containing anti-toxin antibody is placed
perpendicular to the streak of the organism.
Diffusion of the antibody into the medium and
secretion of the toxin into the medium occur.
At the zone of equivalence, a precipitate will form.
Elek plate
 Schick test
Be used to ascertain population risk and
immunization

This test involves the injection of a minute amount of

the diphtheria toxin under the skin. The absence of a
reaction indicates immunity.
Schick test must be done before active immunizatio to know the
persons have been immune or not with diphtheriae.

If persons have been immune with diphtheriae --- continue to Maloney

test to know the persons have been hypersensitive to toxoid

Immunization should be given if the persons were not hypersensitive

to toxoid diphtheriae
Corynebacterium
Guinea pig or tissue culture toxicity assays may
also be done.
Capsule is protein in nature
Cord factor is a complex glycolipid
(trehalose 6,6-dicorynemycolate) that has
been shown to disrupt mouse mitochondria.
It has not been shown to play a role in the
production of diphtheria.
DIAGNOSIS
Clinical: Muscle weakness, edema and a
pseudomembranous material in the upper
respiratory tract characterizes diphtheria.
Laboratory: Tellurite media is the agar of
choice for isolation of Corynebacteria,
which produce jet black colonies
Control
Sanitary: Reduce carrier rate by use
of vaccine.
Immunological: A vaccine (DPT)
prepared from an alkaline formaldehyde
inactivated toxin (i.e. toxoid) is required.
Passive immunization with antitoxin can
be used for patients.
Chemotherapeutic: Penicillin,
erythromycin or gentamicin are drugs of
choice.
Corynebacterium
Clinical Significance (C. diphtheria)
Is normally found in the throats of healthy carriers.
The organism infects only man and it has a limited
capacity to invade.
Diphtheria - Disease usually starts as a local
infection of the mucous membranes causing a
membranous pharyngitis
Local toxin effects result in degeneration of epithelial
cells.
Inflammation, edema, and production of a
pseudomembrane composed of fibrin clots,
leukocytes, and dead epithelial cells and microorganisms
occurs in the throat.
Diphtheria - pseudomembrane
This may obstruct the airway and result in
suffocation.
Corynebacterium
The more dangerous effects occur when the toxin
becomes systemic and attacks the heart
(heart failure), peripheral nerves (paralysis), and
the adrenal glands (hypofunction).
Cutaneous diphtheria More common in tropical
and subtropical areas.
Necrotic lesions with occasional formation of a local
pseudomembrane occur.
Antibiotic susceptibility and treatment
Antiserum once the toxin has bound, however, the
antiserum against it is ineffective.
Penicillin to eliminate the organism.
Corynebacterium
Prevention- Active immunization with toxoid (alum
precipitate).
Is part of the DPT vaccine.
Shick skin test like the Dick test in that it tests for
circulating antibody to the toxin by injecting a
small amount of toxin intradermally and observing
for a local erythematous and necrotic reaction.
If this occurs it indicates that the person has no anti-
toxin antibodies and is, therefore, susceptible to
diphtheria.
Other Corynebacterium are part of the normal
flora of the skin and URT.
Corynebacterium
Are called diphtheroids and may occasionally
cause disease, particularly in immunocompromised
individuals.
C. ulcerans toxigenic strains may produce a disease
similar to, but less severe than diphtheria.
J-K Group commonly cause infections in those with
underlying disease.
Diseases include bacteremia, meningitis, peritonitis, wound
infections, etc.
It is becoming more and more of a problem.
C. pseudotuberculosis found in those with exposure to
animals.
Can cause pneumonia or lymphadenitis.
Produces a different exotoxin than C. diphtheriae.
Transmission and Risk factors

solely among Poor nutrition

humans Crowded or
spread by droplets unsanitary living
secretions conditions
direct contact Low vaccine
coverage among
infants and children
Immunity gaps in
adults
Pathogenesis of diphtheria

Early stages: Sore throat. Low fever.

Swollen neck glands.
Late stages: Airway obstruction and
breathing difficulty. Shock
Prospect

For therapy of tumors !!

tumor
DT

C.Parvum as BRM to tumor

Increase NK/LAK cell activities

Mochammad Hatta: Effect of heat-treated Corynebacterium parvum on hypovolemic shock after acute blood
loss.Proceed. 2nd NATO Civil-Military Blood Conference,Military Hospital Queen Astrid, Brussels, Belgium, 11-13
Mei, 1992, page 25-26.

Mochammad Hatta. Antitumor mechanisms of Eubacterium lentum and Its Components. Asian-Pasific J. Allergy
and Immunology. vol 13: No. 2; 129-137, December (1995)