Diagnostic microbiology

lecture: 13

Gram Positive, Non Endospore-Forming

Bacilli

CORYNEBACTERIUM

Abed ElKader Elottol

MSc. Microbiology

2010

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 Diphtheria and Pertussis 33.3
Corynebacterium diphtheriae •
Spreads by airborne droplets and enters the body –
via the respiratory route
Previous infection or immunization provides –
resistance
Pathogenic strains lysogenized by bacteriophage  –
produce a powerful exotoxin that causes
Tissue death •
The appearance of the pseudomembrane in the •
patient’s throat

.Pearson Education, Inc 2012 ©

 Figure 33.7b

.Pearson Education, Inc 2012 ©

 Diphtheria

Greek diphtheria (leather hide) •

Caused by Aerobic Gram +ve rods •
Corynebacterium diphtheria •
Exotoxin production only if infected by virus •
phage carrying toxin gene

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 Corynebacterium
,Gram + Non Acid fast, Non motile •
,Irregularly stained with granules •
Club shaped swelling at one or both ends so •
the name
Important Pathogen •
,Corynebacterium diphtheria
,Diptheros meaning leather

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 What is Diphtheria
An infection of •
local tissue of URT
with production of
toxin which causes
systemic effects
on Heart and
,Peripheral tissues

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 Definition
Diphtheria is an acute, toxin- •
mediated disease caused by
toxigenic Corynebacterium
.diphtheria
It’s a very contagious and •
potentially life-threatening bacterial
.disease
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 Definition
It’s a localized infectious disease, which 
usually attacks the throat and nose
mucous membrane

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 Etiology
C. diphtheriae is an aerobic gram- •
.positive bacillus
Pleomorphic, club-end –
Non-spore-forming –
Non-acid-fast –
Non-motile –

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 Etiology
The major virulence determinant is an •
exotoxin, diphtheria toxin. After binding
to the host cells, the active subunit will
interrupt the protein synthesis of the
.target host cell and results in cell death
Toxoid made from diphtheria toxin can •
.be used as vaccine

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 Etiology
There are three biotypes — •
gravis, Intermedius, and mitis.
The most severe clinical type of
this disease is associated with
the gravis biotype, but any strain
.may produce toxin
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 Diphtheria Epidemiology
Human carriers Reservoir
Usually asymptomatic

Respiratory Transmission
Skin and fomites rarely

Winter and spring Temporal pattern

Up to several weeks Communicability

without antibiotics

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 Medically Important SPECIES:
1. Corynebacterium diphtheria
2. Corynebacterium ulcerans
3. Corynebacterium ovis (pseudotuberculosis)

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Corynebacterium diphtheria
Clinical manifestation:
•Local lesions in the nose and throat.
•Primary infection may affect ear, conjuctiva, umbilicus and vagina.
•The classic form of the disease is characterized by a local
pseudomembraneous.
•lesion covering the tonsils, pharynx or in the nose.
• The pseudomembrane can extend into trachea and completely
obstruct the air passage, causing the patient to die of suffocation.
•Diphtheria toxin produced in the local lesion which is absorbed
systematically can damage such distant organs and tissues such as
heart, liver, kidneys and central nervous system.
•Death is usually results from heart failure.

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Sign and Symptoms
•The respiratory form has an incubation period of 2-5 days.
•The onset of disease is usually gradual.
• Symptoms include fatigue, fever, a mild sore throat and
problems swallowing.
Children infected have symptoms that include nausea, vomiting,
chills, and a high fever, although some do not show symptoms
until the infection has progressed further.
• In 10% of cases, patients experience neck swelling, informally
referred to as "bull neck."
•These cases are associated with a higher risk of death.
General Characteristics:
•Gram-positive bacilli, Pleomorphic (Curved or straight).
• Non-motile, non-acid fast and aerobic.
•Club-shaped forms = Swelled end
• Chinese characters “Cell arrangement L or V shapes are also
observed ).
•Ability to grow on selective media containing potassium tellurite.
Specimen Collection:
•Throat and nasopharengeal.
• When diagnosis is confirmed, patient contacts must be subjected
to the same procedures.
• Moisten swabs with Normal saline and collect in the usual
manner.

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 Media
Pai Medium
Composition per 1620.0mL:
Glucose...............................................................5.0g
Homogenized whole egg...................................1.0L
Glycerol......................................................120.0mL
pH 6.75 ± 0.2 at 25°C
Use: For the isolation and cultivation of Corynebacterium spp.

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Tinsdale Agar
Composition per 1100.0mL:
peptone............................................................20.0g
Agar..................................................................15.0g
NaCl....................................................................5.0g
Yeast extract........................................................5.0g
L-Cystine...........................................................0.24g
Tinsdale Supplement:
Composition per 100.0mL:
Na2S2O3(Sodium Thiosulphate )................... 0.43g
K2TeO3(Potassium Tellurite) ....................... 0.35g
Serum..........................................................100.0mL
Use: For the primary isolation and identification of Corynebacterium
diphtheriae.
The formation of black to brown halos surrounding the colony results from
the reduction of potassium tellurite to metallic tellurite.

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Loeffler Medium
Composition per liter:
Beef serum........................................................70.0g
Egg, dried............................................................7.5g
Heart muscle, solids from infusion...................0.72g
Glucose.............................................................0.71g
Peptic digest of animal tissue...........................0.71g
NaCl..................................................................0.36g
pH 7.6 ± 0.2 at 25°C
Use: For the cultivation of Corynebacterium diphtheriae. For demonstration of
pigment production and proteolysis by Corynebacterium diphtheriae

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Hoyle Medium Base
Composition per liter:
Agar..................................................................15.0g
Beef extract.......................................................10.0g
Peptone.............................................................10.0g
NaCl....................................................................5.0g
Blood…………....................................................50.0mL
Tellurite solution...........................................10.0mL
pH 7.8 ± 0.2 at 25°C
Tellurite Solution:
Composition per 100.0mL:
K2TeO33.5g................................................................
Caution: Potassium tellurite is toxic.
Use: For the isolation and differentiation of Corynebacterium diphtheriae

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Pai medium and Loeffler medium provides good growth of
C.diphtheria and stained smears prepared from both media may
show the characteristic Chinese letters blue bacillus.
Colony Morphology:
Gray to black with irregular edges on Loeffler medium.
Based on colony morphology Corynebacteria are divided into 3
groups on Tinsdale Agar and Hoyle Medium
1. Gravis: Large, flat, gray to black, non-hemolytic
2. Mitis: Smaller, more convex, darker with dull surface
3. Intermedius: The smallest of all.
Note: Gravis strain hydrolysis starch and glycogen while mitis and
intermedius do not.

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Diphtheria toxin:
1.Structure:
• It is a heat-labile polypeptide that can be lethal in a dose of 0.1
ug/kg.
• If disulfide bond are broken, the molecule can give two
fragments. (A & B).
•Fragment B has no independent action but it is required for the
transport of fragment A.
2. Mechanism of Action:
•Fragment A inhibits polypeptide chain elongation by
inactivating the elongation factor 2 "EF2".
• This factor is required for translocation of polypeptidyl-
transfere RNA from the acceptor to the donor site on the
eukaryotic ribosome.
•Thus preventing protein synthesis leading to cell death.
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Biochemical tests:
The colonial morphology of C. diphtheria on Tinsdale medium is
the most important characteristics in differentiating it from
other species of corynebacteria.
=Urease Negative
=Nitrate Positive
=Gelatin liquefaction: Negative
Fermentation reactions of various carbohydrates.

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Toxigenicity Tests:
1. In-Vivo toxigenicity test:
1. From a pure culture of an isolate, inoculate a 10-ml tube of BHIB,
incubate at 37C for 48 hours.
2. Inject one guinea pig with diphtheria antitoxin to serve as a
control.
3. After 2 hours, inject 5 ml of the broth into both the control and
test animals.
4.Observe after 48 hours.
Results and interpretation
•If the test animal dies and the control survives= It is C. diphtheria
• If both the test and control show illness or die= It is another
organism.

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2. In Vitro toxigenicity test:
Principle: This test is based on a precipitin reaction between
diphtheria toxin produced by the test culture and diphtheria
antitoxin in the medium.
The reaction produces insoluble products that precipitate.
Procedures:
1. Paper strip is saturated with diphtheria antitoxin.
2. The medium contained in a petridish is streaked with the test
organism or organisms if more than one strain are to be tested.
3. Place the paper strip perpendicular to the organism streaks.
4. Incubate the plates for 24 hours at 37 oC.
5. Examine the line of precipitation that extend out from the line
of bacterial growth.
6. The presence of line of precipitation is considered positive
and it confirms that the test organism as a toxigenic.

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Skin test = Schick test
To determine whether The patient is susceptible to diphtheria
infection or not by detecting the presence or absence of
antibodies.
Procedures:
1. 0.1 ml of toxin is injected intradermally in one arm.
2. 0.1 ml of heated (inactivated) toxin as a control is injected
in the other arm.
3. Read after 24-48 hours.
Positive: (Susceptible)Redness and swelling that increases for
several days and then fades, leaving brownish pigmented
area. The control site shows no reaction.
Negative:(Not susceptible) Neither injection site shows any
reaction).

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 Treatment:
1.Antibiotic therapy: Penicillin or erythromycin To
eradicate the organism.
2. Anti-toxin: To neutralize the effect of diphtheria toxin.
Control:
By immunization with inactivated or attenuated toxin of
toxigenic C.diphtheria.

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 Erysipelothrix insidiosa
E. Rhusiopathiae

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 Disease: Erysipleoid (red skin)
it has three distinct clinical entities:
1. Erysipeloid : Localized cutaneous infection usually on the
finger and hands.
2. Generalized cutaneous infection.
3. Septicemia
Microorganism:
- Gram-positive rods.
- Non-motile.
- Non-spore forming.
- Facultative anaerobe.

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 Specimen and media:
• Usually skin biopsy from the advancing borders of the lesion.
• Blood specimen if septicemia is suspected.
•-Culture media: 1% glucose broth and blood agar.
NB: Make gram staining for the organism from the clinical
specimen and the growth on blood agar.
Biochemical tests used in the identification:
•Test tube brush appearance in gelatine stabs.
• H2S production.
• Acidification of slant and butt in TSIA with no gas production.
• To differentiate this organism from Listeria monocytogenes,
perform the catalase test. Listeria monocytogenes is catalase
Positive.
Treatment:
- Penicillin is the drug of choice.

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 Listeria monocytogenes

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 Disease: Listerosis
= it has four forms
1. Non-specific flu-like illness during pregnancy or
puerperal sepsis.
2. Neonatal sepsis and later meningitis.
3. Sepsis or meningitis in immunocompromised patients.
4. Food poisoning.
Microorganism:
- Gram-positive rods (Shorter & Thinner).
- Non-spore forming
- Facultative anaerobe
- Beta hemolytic on Blood agar.

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Biochemical Characteristics:
 Motile (Umbrella-type growth in semi-solid media)
 Motile (Tumbling motility as seen by the hanging drop
technique)
 Catalase positive
 V.P positive
Specimen and Culture media:
Blood, cerebrospinal fluid (CSF) and genital tract secretion.
Media : BHIA + 5% sheep blood and BHIB.
 Selective Listeria Agar SLA
= Brownish to black discoloration is usually seen around the
colonies of Listeria monocytogenes.

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 Identification:
1. The demonstration of Gram-positive bacilli from stained
smears (CSF and other fluids or food samples).
2. Culture and Isolation: On Blood Agar or Selective Listeria
Agar.
3. Biochemical tests: Catalase test, V.P, and motility test.
4. Serological test: Agglutination with specific sera.

Treatment:
Combined therapy of penicillin plus aminoglycosides.
Tetracycline also may be used as a second line if the patient is
sensitive to penicilllin or aminoglycosides.

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 The same Blood Agar plate examined with
Pinpoint to small, semi-transparent
transmitted light. The colonies are surrounded
.colonies
by a narrow haemolytic zone. This zone is
more easily seen if the culture is grown on thin
Blood Agar plates.
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 Listeria monocytogenes grows as brown-green colored
colonies with a black halo (esculin splitting).

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 END of LECTURE

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