Diphtheria - Prac. Microbiology

Gram positive bacilli
Non-spore forming
- Corynebacterium
- Listeria
Spore- forming
- Bacillus
- Clostridium

Non-spore forming
Corynebacterium

Corynebacterium
 Some species are part of normal flora of
skin and mm.
 Medically important species is
Corynebacterium diphtheriae

Morphology
 Gram-positive bacilli
 Club-shaped
 Arranged at acute angles or
parallel to each other
(Chinese letters).
 Meta-chromatic granules.
 Non-spore forming

Methylene blue stain:
Beaded appearance

Culture Characters
 Aerobic.
 Growth on:
1. Blood agar
2. Loeffler’s serum:
Best morphology
3. Blood tellurite agar:
Selective & differential
Grey to black colonies

Virulence factors
Diphtheria ExotoxinDiphtheria Exotoxin
Exotoxin is dependent on:
1. Lysogenic prophage.
2. Low extracellular iron
concentration.

Disease: Diphtheria
 Upper respiratory tract infection.
 Transmitted by droplets.
 Characterized by:
1- Local pseudomembrane.
2- Toxemia.
 Complications:
 Airway obstruction
 Toxic myocarditis and heart failure
 Nerve paralysis

Clinical Manifestations
- Cervical lymphadenitis
(Bull neck)
- Toxaemia with low grade fever
D.D. of sore throat:
1- S. pyogenes
2- Vincent’s angina
3- C. diphtheriae

Diagnosis
Mainly clinical
Laboratory confirmation:
A- Specimen:
Throat swab from the
pseudomembrane.

B- Direct Detection:
Microscopic examination (Gram stain):
• Gram-positive bacilli
• Chinese letters appearance

B- Direct Detection:
Microscopic examination (Methylene Blue stain):
Meta-chromatic granules

1- Loeffler’s serum:
Best morphology
2- Blood tellurite agar:
grey/black colonies
3- Blood agar to
exclude S. pyogenes
C- Cultivation:

D- Identification:
Microscopic examination:
1- Gram stained smear:
Gram-positive club-shaped
bacilli (Chinese letters).
2- MB stained smear: showing
meta-chromatic granules.

The isolated organism is
Corynebacterium diphtheriae
Is it Toxigenic or Not?

E- Toxigenicity Tests:
a) Elek’s test: most common assay.
b) PCR: detection of toxin gene.
c) ELISA: detection of toxin from culture.

Elek’s test:
An antigen-antibody reaction in which the
Ag is soluble “Precipitation”.

Diagnosis of carriers
Throat or nasal swabs are subjected to
the same procedures:
Isolation
+
Toxigenicity tests

What treatment is prescribed?
 Treatment should be IMMEDIATELY started
if diphtheria is clinically suspected.
 Diphtheria antitoxin and antibiotics.
 Treatment of symptoms & complications
e.g. respiratory support.

How can we prevent this disease?
By Vaccination
Diphtheria toxoid + pertussis vaccine +
tetanus toxoid in a trivalent vaccine:
DPT
For close contacts of a case:
(booster of diphtheria toxoid + antibiotic
chemoprophylaxis)

Diphtheroids
Corynebacteria that resemble C.diphtheriae in
morphology.
They are mainly commensals.

Case
 A 4-year-old male child
presented with fever of 38°C.
 Physical examination
revealed clear chest,
exudative pharyngitis and
bilaterally enlarged cervical
lymph nodes.
 A throat culture was taken
and a course of penicillin was
started.

Case (cont.)
 The child’s course worsened, he
became increasingly lethargic,
developed respiratory distress and
was hospitalized.
 On admission, he had a fever of
38°C and an exudate in the
posterior pharynx described as a
yellowish, thick membrane which
bled when scraped and removed.
 The patient’s medical history
revealed that he had received no
immunizations.

Listeria monocytogenes
Gram-positive
rods
(coccobacilli)
Microscopic examination:

Listeria resembles Corynebacteria in
morphology but is MOTILE.

Diseases
 Abortion, premature delivery or sepsis
during the peripartum period.
 Neonatal meningitis
 Septicaemia and meningitis (in
immunocompromised adults).
 Food poisoning (dairy products or
undercooked meat)

Neonatal meningitis
 Meningitis caused by Listeria is almost
always seen in neonates.
 Causes of Neonatal Meningitis:
1. Group B Streptococci
2. E. coli K1
3. Listeria monocytogenes

Case
A one month old girl was admitted to hospital
with acute meningitis.
The Gram stain of CSF revealed Gram-positive
short rods.
What is the cause of neonatal meningitis?
a. N. meningitidis, group A
b. N. meningitidis, group C
c. Listeria monocytogenes
d. S. pneumoniae

How did the mother contract it?
 Listeriosis is a food-borne infection.
 Listeria resists drying, heating and freezing
without forming spores.
 Commonly contaminated food items:
1. Dairy products (esp. unpasteurized milk and
soft cheeses).
2. Undercooked meat (chicken, hot-dogs).
3. Refrigerated food.

1- C.diphtheriae is cultured on:
a- Nutrient agar.
b- Chocolate agar.
c- Loffler’s serum.
d- Lowenstein-Jensen medium.
e- MacConkey’s agar.

2- Blood tellurite agar is a(n):
a- Enriched medium
b- Enrichment medium
c- Simple medium
d- Selective and differential medium
e- Indicator medium only

3- Which of the following is a toxigenicity test
for C.diphtheriae ?
a- Elek’s test
b- Coagulase
c- Catalase test
d- Culture on blood tellurite
e- ELISA test for antibody detection

4- C. diphtheriae has the following morphology:
a- Gram negative cocci arranged in pairs
b- Gram positive cocci arranged in chains
c- Gram positive club-shaped bacilli
d- Gram positive cocci in clusters
e- Gram positive capsulated diplococci

5- The toxin of C. diphtheriae is only produced
by those strains that are:
a- Encapsulated.
b- Glucose fermenters.
c- Sucrose fermenters.
d- Lysogenic
e- Endotoxin producers.

6- A 1-week old newborn develops meningitis.
Short gram-positive rods are isolated. The
mother had eaten unpasteurized cheese during
pregnancy. What is the most likely etiological
diagnosis?
a- C. diphtheriae.
b- S. pyogenes.
c- L. monocytogenes.
d- S. pneumoniae.
e- S. agalactiae

Diphtheria - Prac. Microbiology

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