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Lec-5 Genus Corynebacterium د.انسام محمد
Non spore forming gram positive rods
The genus Corynebacterium comprises 66 species, 38 of them are of clinical significance.
The majority are normal flora on the skin and mucous membrane of human and animals.
They frequently show club-shaped swellings and hence the name Corynebacteria (from
coryne, meaning club).
The term dephtheroid means diphtheria like. The most significant pathogen of the group is
C. diphtheriae. Other spp. are C.bovis, C.ulcerans, C.xerosis, C.jekeium etc.
Corynebacterium diphtheriae
Morphology
They are, thin, gram-positive bacilli, highly pleomorphic, non-motile and non-spore
forming with a tendency to clubbing at one or both ends, arranged in pairs, palisades or
resembling the letters V or L. This particular arrangement is called the Chinese letter
arrangement.
This organism has granular and uneven staining. These granules are known as
metachromatic granules, volutin granules or Babes Ernst granules which are often
situated at the poles of the bacilli and are called polar bodies. Special stain; Albert’s stain
has been used for demonstrating the granules clearly. The granules represent accumulation
of polymerized polyphosphates.
Cultural Characteristics
C. diphtheriae is a facultative anaerobe, It can grow on ordinary nutrient agar. The growth
is improved on enriches media. The media are useful for this purpose:
1. Loeffler’s serum slope 2. Tinsdale agar
3. Tellurite blood agar: The addition of potassium tellurite makes the medium selective for
Corynebacteria by inhibiting most other pathogenic and commensal bacteria. On this
medium, C. diphtheriae give grey/black, shiny or dull colonies.
Based on colonial morphology on the tellurite medium and other properties, there are
four biotypes of C diphtheriae have been widely recognized: gravis, mitis, intermedius,
and belfanti. They are classified on the basis of growth characteristics such as colony
morphology, biochemical reactions, and severity of disease produced by infection.
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Toxin
Only toxin producing C. diphtheriae causes the disease diphtheria. Toxigenic strains of C.
diphtheriae produce a very powerful exotoxin. The toxigenicity of the C. diphtheriae
depends on the presence of corynephages (tox+), which act as the genetic determinant
controlling toxin production. Non-toxigenic strains can be converted to tox+ by infection
with the appropriate bacteriophage. This is known as lysogenic or phage conversion.
When some non-toxigenic diphtheria organisms are infected with bacteriophage from
certain toxigenic diphtheria bacilli, the offspring of the exposed bacteria are lysogenic and
toxigenic, and this trait is subsequently hereditary.
The toxin consists of two fragments A (active) and B (binding). Both fragments are required
for toxicity. Fragment A has all the enzymatic activity whereas fragment B is responsible for
binding the toxin to the cells and mediates the entry of fragment A into the cytoplasm. The
toxin is heat labile. It has a special affinity for certain tissues such as the myocardium,
adrenals and nerve endings.
This toxin is very potent and 130ng/Kg is lethal for humans. The production of the toxin in
vitro needs: a. alkaline PH. b. oxygen. c. low iron.
Mode of Action
The diphtheria toxin acts by inhibiting protein synthesis which is responsible for both the
necrotic and neurotoxic effects of the toxin.
The “virulence” of diphtheria bacilli is attributable to their capacity for establishing
infection, growing rapidly, and then quickly elaborating toxin that is effectively absorbed.
Clinical infections
The organism is carried in the upper respiratory tract and spread by droplet infection or
hand-to-mouth contact. The incubation period of diphtheria is 2–5 days. It occurs in two
forms (respiratory and cutaneous) and is found worldwide.
A. Respiratory Diphtheria
The illness begins gradually and is characterized by low-grade fever, malaise, and a mild
sore throat. The most common site of infection is the tonsils or pharynx. The organisms
rapidly multiply on the epithelial cells, and the toxigenic strains of C. diphtheriae produce
toxin locally, causing inflammatory reaction, tissue necrosis and exudate formation
forming a tough gray to white pseudomembrane, which attaches to the tissues commonly
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over the tonsils, pharynx, or larynx. Any attempt to remove the pseudomembrane results
in bleeding.
In nasopharyngeal infection, the pseudomembrane may involve nasal mucosa, the
pharyngeal wall and the soft palate. In this form, oedema involving the cervical lymph
glands may occur in the anterior tissues of the neck, a condition known as bull-neck
diphtheria.
Laryngeal involvement leads to obstruction of the larynx and lower airways.
The toxin also is absorbed and can produce a variety of systemic effects involving the
kidneys, heart, and nervous system, although all tissues may be affected.
Intoxication takes the form of myocarditis and peripheral neuritis, and may be associated
with thrombocytopenia. Difficulty in swallowing and paralysis of the arms and legs also
occur but usually resolve spontaneously. Death is most commonly due to congestive heart
failure and cardiac arrhythmias.
B. Cutaneous Diphtheria
It is prevalent in the tropics, the toxin also is absorbed systemically, but systemic
complications are less common than from upper respiratory infections.
Laboratory Diagnosis
Specific treatment should be done immediately on suspicion of diphtheria without waiting
for laboratory tests. Any delay may be fatal. Laboratory diagnosis consists of isolation of
the diphtheria bacillus and demonstration of its toxicity.
1. Specimens
Swabs from the nose, throat, or other suspected lesions must be obtained before
antimicrobial drugs are administered.
2. Microscopy
Direct microscopy of a smear is unreliable since C. diphtheriae is morphologically similar to
other coryneforms. Stained smears show beaded rods in typical arrangement.
3. Culture
The swab should be inoculated on Loffler’s serum slope, tellurite blood agar, and blood
agar. The cultures should be incubated aerobically at 37°C.
4. Virulence Tests
To test for toxigenicity of an isolated diphtheria. Diagnosis of diphtheria
depends on showing that the isolate produces diphtheria toxin.
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A. In vivo tests
i. Subcutaneous test: The growth from an overnight culture on Loeffler’s slope is
emulsified in 2–4 ml broth and 1 ml of the emulsion injected subcutaneously into two
guinea pigs or rabbits, one of which has been protected with the diphtheria antitoxin (500–
1000 units) 18–24 hours previously and was used as control. If the strain is virulent, the
unprotected animal will die within four days.
ii. Intracutaneous test no loss of the animal.
B. In vitro Test
i. Elek’s gel precipitation test: The in vitro diphtheria toxin detection procedure is an
immunodiffusion test first described by Elek.
Procedure: A rectangular strip of filter paper impregnated with diphtheria antitoxin (1000
units/ ml) is placed on the surface of a 20% normal horse serum agar in a Petri dish while
the medium is still fluid. When the agar has set, the surface is dried. The plate should be
streaked with the test strain as well as the control positive and negative strains at right
angles to the strip in a single straight line and parallel to each other. The plate is incubated
at 37°C and examined after 24 and 48 hours.
Interpretation: Toxins produced by the bacterial growth will diffuse in the agar and where
it meets the antitoxin at optimum concentration will produce a line of precipitation. A
negative control should be free of any line. No precipitate will form in the case of non-
toxigenic strains.
ii. Tissue culture test: The toxigenicity of diphtheria bacilli can be demonstrated by
incorporating the strains in the agar overlay of cell culture monolayers. The toxin produced
diffuses into the cells below and kills them.
iii. Enzyme-linked immunosorbent assays (ELISA): are available for the detection of
diphtheria toxin.
iv. Polymerase chain reaction (PCR): for detecting the C. diphtheriae tox gene. The PCR
assay can also be applied directly to clinical specimens.
Prophylaxis
The methods of immunization available are active, passive or combined.
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A. Active Immunization
1. Single vaccines are less frequently used
2. Combined preparations:
– DPT (diphtheria-pertussis-tetanus) vaccine trivalent preparation
B. Passive Immunization
This is an emergency measure to be employed where susceptible (non-immunized) are
exposed to infection. It consists of the subcutaneous administration of 500–1000 units of
antitoxin (antidiphtheritic serum, ADS).
Treatment
Specific treatment of diphtheria consists of antitoxic and antibiotic therapy. Antitoxin
should be given immediately as soon as clinical diagnosis is made to neutralize the toxin
being produced. The dosage recommended is 20,000 units intramuscularly for moderate
cases and 50,000 to 100,000 units for serious cases, half the dose being given intravenously.
C. diphtheriae is sensitive to most antibiotics, including penicillin and erythromycin for the
treatment of patients as well as carriers. The antibiotics do not neutralize circulating toxin.
Penicillin-sensitive individuals can be given erythromycin. Erythromycin is more active than
penicillin in the treatment of carriers.
Diphtheroids
Corynebacteria resembling C. diphtheriae occur as normal commensals in the throat, skin
and other areas. These may be mistaken for diphtheria bacilli and are known as
diphtheroids. They can be differentiated from C. diphtheria on the basis of biochemical
characters and toxigenicity tests. The common diphtheroids are C. pseudodiphtheriticum
and C. xerosis.
Listeria monocytogenes
L.monocytogenes is an important human pathogen, wide spread in the environment
recovered from soil, water, and animal products. It has tropism to the CNS. L
monocytogenes is capable of growing and surviving over a wide range of environmental
conditions. It can survive at refrigerator temperatures (4°C), under conditions of low pH
and high salt conditions. Therefore, it is able to overcome food preservation and safety
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barriers, making it an important food-borne pathogen. It
secretes
Listeriolysin O which
damages the phagosome membrane and inhibit killing by macrophages
Clinical infections
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Pregnant women: flulike illness with fever head ache and myalgia, seen mostly in the
third trimester. It may cause premature labor, septic abortion within 3-7 days, spontaneous
abortion and stillborn neonates. The infection usually self limited.
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Newborn: causes serious infections. Early onset associated with aspiration of
infected amniotic fluid and have high mortality rate. Late onset disease occurs several days
or weeks after birth with lower fatality rate. The disease manifest as meningitis.
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Immunosuppressed host: invasive listeriosis causing CNS infection with high fatality
rate.
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Healthy people: cause GIT infection when they eat food contaminated with the MO.
Laboratory diagnosis
Microscopically: gram positive coccobacilli, singly, short chains or palisades.
Culture: grow on SBA and chocolate agar as well as nutrient agar. The colonies are small
round smooth and translucent surrounded by narrow zone of beta hemolysis. The MO
grows at 4C° so cold enrichment is used to isolate the MO using broth incubated at 4C° for
several weeks.
Identification: catalase positive, motile, beta hemolytic, CAMP test positive.
References
1-
Koneman's color atlas and textbook of diagnostic microbiology 7
th
edition, 2017.
2-
Bailey and Scott's Diagnostic Microbiology 14
th
edition, 2017.
3-
Jawetz, Melnick and Adelberg's medical microbiology 26
th
edition, 2013.