Corynebacterium diphteriae

3 RD YEAR PHARMACY, SEMESTER 2, 2021

DR. HENG KANIKA,PHARMD
Learning objectives
At the end of this session students should be able to:
➢ Describe morphology of Corynebacterium diphteriae
➢ Describe epidemiology of Corynebacterium diphteriae
➢ Explain the pathogenesis of Corynebacterium diphteriae
➢ Cite the clinical signs and symptoms given by Corynebacterium diphteriae
➢ Describe the diagnostic methods to identify bacteria
➢ Describe Prevention and treatment of Diphteria
 Content
◦ Introduction
◦ Epidemiology
◦ Pathogenesis
◦ Clinical signs and Symptoms
◦ Diagnosis
◦ Treatment and Prevention
 Introduction
Pathogenic bacterium that causes diphtheria. It is also known as the Klebs-
Löffler bacillus, because it was discovered in 1884 by
German bacteriologists Edwin Klebs (1834–1912) and cultivated by Friedrich
Löffler (1852–1915).
Four subspecies are recognized:
Corynebacterium diphteriae mitis,
Corynebacterium diphteriae intermedius,
Corynebacterium diphteriae gravis,
Corynebacterium diphteriae belfanti
 Introduction
• Gram-positive pleomorphic rods
•Thick peptidoglycane cell wall
• Club shaped due to the presence of metachromatic (volutin) granules at one or
both ends.
• Chinese letter or cuneiform arrangement
•Non motile
• Aerobic bacteria
• Non spore forming Stained Corynebacterium cells. The "barred"
appearance is due to the presence of
polyphosphate inclusions called metachromatic
granules. Note also the characteristic "Chinese-
letter" arrangement of cells.
 Introduction
Fastidious organisms; grows best at 37 °C on blood or serum-containing media
such as Loeffler’s medium, tellurite medium, etc
The major virulence factor is the diphtheria toxin, an A-B exotoxin; inhibits
protein synthesis
Etiologic agent of diphtheria: respiratory and cutaneous forms
 Epidemiology
Worldwide distribution maintained in asymptomatic carriers and infected
patients
Humans are the only known reservoir, with carriage in oropharynx or on skin
surface
Spread person to person by exposure to respiratory droplets or skin contact
Disease observed in unvaccinated or partially immune children or adults
traveling to countries with endemic disease
Diphtheria is very uncommon in the United States and other countries with
active vaccination programs
 Epidemiology
Risk factors for the spread of diphtheria include:
• ​Overcrowded areas
• Poor hygiene
• Lack of immunisation
 Pathogenesis
Diphtherial infections are mainly because of the toxin.
The virulence of Diphtheria bacilli is due to capacity to
◦ Establish infection and grow rapidly
◦ Quickly elaborate an exotoxin
Diphtheria toxin is an exotoxin secreted by C. diphtheria. Diphtheria
toxin is a single polypeptide chain of 535 amino acids consisting of
two subunits linked by disulfide bridges, known as an A-B toxin.
 Pathogenesis
There are at least four main steps involved in intoxication of a single eukaryotic
cell by diphtheria toxin:
(1) the binding of the toxin to surface receptor of its target cell;
(2) grouping of charged receptors into layered pits and internalization of the
toxin by receptor-mediated endocytosis; followed by acidification of the
endocytic vesicle by a membrane-associated, ATP-driven proton pump,
(3) Insertion of the transmembrane domain (B-subunit) into the membrane and
smoothed the delivery of catalytic domain (A-subunit) to the cytosol,
(4) the ADP-ribosylation of elongation factor 2 (EF-2), which results in the
permanent inhibition of protein synthesis. A single molecule of the catalytic
domain delivered to the cytosol is enough to be deadly for the cell.
Pathogenesis
 Pathogenesis
C. diphtheriae occurs in the respiratory tract, in wounds, or on the skin of
infected persons or normal carriers. It is spread by droplets or by direct contact.
Portal of entry: respiratory tract or skin abrasions. Diphtheria bacilli colonize and
grow on mucous membranes, and start to produce toxin, which is then absorbed
into the mucous membranes, and even spread by the bloodstream.
Local toxigenic effects: elicit inflammatory response and necrosis of the facial
mucosa cells-- formation of "pseudo-membrane“ (composed of bacteria,
lymphocytes, plasma cells, fibrin, and dead cells), causing respiratory
obstruction.
Systemic toxigenic effects: necrosis in heart muscle, liver, kidneys and adrenals.
Also produces neural damage.
Clinical signs and Symptoms
 Diagnosis
Sample: Swabs (preferably two) from the lesion of throat, larynx or nasal cavity; one
for direct examination and another for culture or a portion of the pseudomembrane.
Direct examination
•Smears of the throat swab should be stained with both Gram stain and methylene
blue or Albert stain.
. Culture
• Loeffler’s medium:
best morphology
 Diagnosis
Blood tellurite agar: It is a selective and differential medium
for C. diphtheriae.After 48-72 hours, colonies of C. diphtheria
appear as small, grey, or black with a raised center.
Tinsdale’s Agar: After incubation for at least 48 hours, colonies
of Corynebacterium diphtheriae appear black with dark brown halos

Rapid identification methods

API Coryne strip and RapID CB Plus are commercial products available for the rapid identification
of Corynebacterium diphtheriae.
 Diagnosis
Toxigenicity testing
• Elek immunodiffusion test: It is the most common in vitro assay for determining toxigenicity of C.
diphtheriae. This test is based on the double diffusion of diphtheria toxin and antitoxin in an agar
medium. A sterile, antitoxin-saturated filter paper strip is embedded in the culture medium, and C
diphtheriae isolates are streak-inoculated at a 90° angle to the filter paper. The production of
diphtheria toxin can be detected within 18 to 48 hours by the formation of a toxin-antitoxin precipitin
band in the agar.
 Diagnosis
• Detection of toxin gene by polymerase chain reaction (PCR).
• In vivo test: inject the culture into antitoxin-protected and unprotected guinea pigs
subcutaneously.
 Treatment
Penicillin
Erythromycin
Anti toxin from horse serum to stop the toxin made by the bacteria from damaging the
body. This treatment is very important for respiratory diphtheria infections, but it is rarely used
for diphtheria skin infections.
 Prevention
Diphtheria vaccine is a bacterial toxoid, a toxin whose toxicity has been
inactivated.
The vaccine is normally given in combination with other vaccines as DTwP/DTaP
vaccine or pentavalent vaccine. For adolescents and adults the diphtheria toxoid
is frequently combined with tetanus toxoid in lower concentration (Td vaccine).
CDC is recommended that children receive 5 doses of DTaP, usually at the
following ages:
◦ 2 months
◦ 4 months
◦ 6 months
◦ 15–18 months
◦ 4–6 years
 References
◦ P.Murray-Medical Microbiology ,7th edition,2012
◦ Diphteria - CDC Fact Sheet (Detailed)
◦ www.osmosis.org/learn/Corynebacterium diphtheria
◦ https://www.who.int/immunization/diseases/diphtheria/en/