Lecture 4
 Gram positive bacilli, with characteristic
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morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Facultative anaerobic
Breakdown glucose by oxidative and fermentative
i.e. O+/F+
C. diphtheriae is fastidious while diphtheriods are
non-fastidious
Catalase positive
Oxidase negative
Corynebacterium
It caused
diphtheria
Pathogenic
C. diphtheriae
C. diphtheriae is the only
pathogenic members of this
genus
Commensal
"Diphtheriods"
C. hofmannii, C.
xerosis, C. acne
Normal flora of RT, urethra,
vagina, Skin
Gram Positive Bacilli
Gram positive rods
Spore forming
Aerobic
Bacillus spp
Non spore forming
Anaerobic
Clostridium spp
Corynebacterium
 The non-spore-forming gram-positive
bacilli are a diverse group of bacteria.
Many members of the genus
corynebacterium and their anaerobic
equivalents, propionibacterium
species, are members of the normal
flora of skin and mucous membranes of
humans.
Corynebacterium diphtheriae is the
most important member of the group, as
it can produce a powerful exotoxin that
causes diphtheria in humans. Listeria
monocytogenes
and Erysipelothrix rhusiopathiae are
primarily found in animals and
occasionally cause severe disease in
humans
 Based upon Gram stain morphology,
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formation of spores, and catalase reaction
Spore-forming, catalase positive (aerobic):
Bacillus sp.
Regularly-shaped, non-spore forming
Catalase positive: Listeria monocytogenes
Catalase negative: Erysipelothrix
rhusiopathiae, Lactobacillus sp., Gardnerella
vaginalis
Irregularly-shaped, non-spore forming,
catalase positive: Corynebacterium sp.
(“diphtheroids”)
Branching: Nocardia sp., Actinomyces sp., &
Streptomyces sp.
 Corynebacterium species “coryneform
bacteria” have a high guanosine plus
cytosine content and include the genera
corynebacterium, arcanobacterium,
brevibacterium, mycobacterium, and
others.
 Actinomyces and propionibacterium
are classified as anaerobes, but some
isolates grow well aerobically
(aerotolerant) and must be
differentiated from the aerobic
coryneform bacteria.
Other nonspore- forming gram-positive
bacilli have more regular shapes and a
lower guanosine plus cytosine content.
The genera include listeria and
erysipelothrix; these bacteria are more
closely related to the anaerobic
lactobacillus species, which sometimes
grow well in air, to the sporeforming
bacillus and clostridium species—and
to the gram-positive cocci of the
staphylococcus and streptococcus
species—than they are to the
coryneform bacteria.
 There is no unifying method for
identification of the gram-positive
bacilli. Few laboratories are equipped to
measure guanosine plus cytosine
content. Growth only under anaerobic
conditions implies that the isolate is an
anaerobe, but many isolates of
lactobacillus, actinomyces, and
propionibacterium species and others
are aerotolerant.
 Corynebacteria are 0.5–1 μm in diameter
and several micrometers long.
Characteristically, they possess irregular
swellings at one end that give them the
“clubshaped” appearance.
Irregularly distrib uted within the rod
(often near the poles) are granules
staining deeply with aniline dyes
(metachromatic granules)
that give the rod a beaded appearance.
On blood agar, the C diphtheriae
colonies are small, granular, and gray,
with irregular edges, and may have small
zones of hemolysis. On agar containing
potassium tellurite, the colonies are
brown to black with a brownblack
halo because the tellurite is reduced
intracellularly (staphylococci and
streptococci can also produce black
colonies).
C diphtheriae and other corynebacteria
grow aerobically on most ordinary
laboratory media. Propionibacterium is
an anaerobe. On Loeffler’s serum
medium, corynebacteria grow much
more readily than other respiratory
organisms, and the morphology of
organisms is typical in smears.
Pathogenesis
The principal human pathogen of the
group is C diphtheriae. In nature, 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 contact to
susceptible individuals; the bacilli then
grow on mucous membranes or in skin
abrasions, and those that are toxigenic
start producing toxin.
All toxigenic C diphtheriae are capable of
elaborating the same disease-producing
exotoxin. In vitro production of this toxin
depends largely on the concentration of
iron. Toxin production is optimal at 0.14 μg
of iron per milliliter of medium but is
virtually suppressed at 0.5 μg/mL. Other
factors influencing the yield of toxin in vitro
are osmotic pressure, amino acid
concentration, pH, and availability of
suitable carbon and nitrogen sources. The
factors that control toxin production in vivo
are not well understood.
 Diphtheria toxin is a heat-labile
polypeptide that can be lethal in a dose
of 0.1 μg/kg.
Pathology
 Diphtheria toxin is absorbed into the
mucous membranes
 and causes destruction of epithelium
and a
 superficial inflammatory response. The
necrotic epithelium
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becomes embedded in exuding fibrin and red and
white cells, so that a grayish “pseudomembrane” is
formed—commonly over the tonsils, pharynx, or larynx.
Any attempt to remove the pseudomembrane
exposes and tears the capillaries and thus results in
bleeding. The regional lymph nodes in the neck enlarge,
and there may be marked edema of the entire neck. The
diphtheria bacilli within the membrane continue to
produce toxin actively. This is absorbed and results in
distant toxic damage, particularly parenchymatous
degeneration, fatty infiltration, and necrosis in heart
muscle, liver, kidneys, and adrenals, sometimes accompanied
by gross hemorrhage. The toxin also produces
nerve damage, resulting often in paralysis of the soft
palate, eye muscles, or extremities
Diagnostic Laboratory Tests
Swabs from the nose, throat, or other
suspected lesions must be obtained
before antimicrobial drugs are
administered. Smears stained with
alkaline methylene blue or Gram’s stain
show beaded rods in typical
arrangement.
Laboratory diagnosis of case
 Specimen: A throat swap
 Culture: The swap is inoculated on
Loeffler's serum medium and/or on
blood tellurite agar aerobically at 37C
for 24.
 On Loeffler's serum medium:
 Corynebacteria grow much more
readily than other respiratory
pathogens
 Deep blue or red metachromatic
granules (accumulated inorganic
polyphosphates) by methylene blueLoefflers serum
stain
The colonies of C. diphtheriae are small, granular,
grey, smooth, and creamy with irregular edges
 On blood tellurite agar
 It is selective medium for isolation of C. diphtheriae (Potassium
tellurite)
 3 biotypes of C. diphtheriae are characterized on BTA
 i.e. Gravis, mitis and intermedius biotypes
 The most severe disease is associated with the gravis
biotype
 Colony of gravis biotype is large, non-hemolytic & grey.
 Colonies of mitis biotype are small, hemolytic and black
 Colonies of intemedius biotype are intermediate in size,
non-hemolytic with black center & grey margin.
 All Corynebacterium species are catalase
positive (Also, Staphylococcus and Bacillus
species are catalase positive)
2- Carbohydrate Fermentation Test:
Principle:
 Each species of corynebacteria has its specific
carbohydrate fermentation pattern
 C.diphtheriae can be differentiated from
other Corynebacterium species by
fermentation of glucose and maltose but not
sucrose with production of acid only
Procedure:
Inoculate three tubes of carbohydrate
fermentation medium (broth containing
one type of sugar and phenol red as the pH
indicator) with the test organism
 Incubate the tubes at 37oC for 24 hrs.
Glucose
Maltose
Sucrose
2- Carbohydrate Fermentation Test:
Principle:
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Each species of corynebacteria has its specific carbohydrate fermentation
pattern
C.diphtheriae can be differentiated from other Corynebacterium species by
fermentation of glucose and maltose but not sucrose with production of acid
only
Procedure:
 Inoculate three tubes of carbohydrate
fermentation medium (broth containing
one type of sugar and phenol red as the pH
indicator) with the test organism
 Incubate the tubes at 37oC for 24 hrs.
Glucose
Maltose
Sucrose
Results:
Sugar fermentation can be indicated by change of color of the medium
from red to yellow due to formation of acid which decrease the pH
Glucose
+ve
Maltose
+ve
C. xerosis
Sucrose
+ve
Glucose
+ve
Maltose
+ve
C. diphtheriae
Sucrose
- ve
 Principle:
 It is toxin/antitoxin reaction
 Toxin production by C.diphtheriae can be demonstrated by a
precipitation between exotoxin and diphtheria antitoxin
 Procedure:
 A strip of filter paper impregnated with diphtheria
antitoxin is placed on the surface of serum agar
 The organism is streaked at right angels to the filter
paper
 Incubate the plate at 37C for 24 hrs
Filter paper saturated with diphtheria antitoxin
 Resuls:
Lines of precipitations
 After 48 hrs incubation, the
antitoxin diffusing from filter
paper strip and the toxigenic
strains produce exotoxin, which
diffuses and resulted in lines
four precipitation lines
radiating from intersection of
the strip and the growth of
organism
Inoculated M.O.
Positive Elek’s Test
Resistance & Immunity
Since diphtheria is principally the result
of the action of the toxin formed by the
organism rather than invasion
by the organism, resistance to the disease
depends largely on the availability of
specific neutralizing antitoxin in the
bloodstream and tissues. It is generally
true that diphtheria occurs only in persons
who possess no antitoxin (or less than 0.01
Lf unit/mL). Assessment of immunity to
diphtheria toxin for individual patients can
best be made by review of documented
diphtheria toxoid immunizations and
primary or booster immunization if
needed
The treatment of diphtheria
rests largely on rapid suppression
of toxin-producing bacteria by
antimicrobial drugs and the early
administration of specific
antitoxin against the toxin
formed by the organisms at their
site of entry and multiplication.
Antimicrobial drugs (penicillin,
erythromycin) inhibit the growth of
diphtheria bacilli. Although these
drugs have virtually no effect on the
disease process, they arrest toxin
production. They also help to eliminate
coexistent streptococci and C diphtheriae
from the respiratory tracts of patients or
carriers.
 Propionibacterium species) reside in
normal skin. Propionibacterium acnes,
is aerotolerant and grows aerobically. It
participates in the pathogenesis of acne
 Arcanobacterium haemolyticum produces
betahemolysis on blood agar. It is
occasionally associated with pharyngitis
LISTERIA MONOCYTOGENES
There are several species in the genus
listeria. Of these, L monocytogenes is
important as a cause of a wide spectrum
of disease in animals and humans.
Culture & Growth Characteristics Listeria
grows on media such as Mueller-Hinton
agar.
Identification is enhanced if the primary
cultures are done on agar containing sheep
blood, because the characteristic small
zone of hemolysis can be observed around
and under colonies. Isolation can be
enhanced if the tissue is kept at 4 °C for
some days before inoculation bacteriologic
media.
The organism is a facultative anaerobe and
is catalase-positive and motile. Listeria
produces acid but not gas in a variety of
carbohydrates.
The motility at room temperature and
hemolysin production are primary findings
that help differentiate listeria from
coryneform bacteria.
ACTINOMYCETES
Actinomycetes are a large, diverse group of
gram-positive bacilli with a tendency to
form chains or filaments.
They are related to the corynebacteria and
mycobacteria as well as the stepomycete
 The most famous species are
 Actinomyces israelii
 Actinomyces naeslundii
 Diagnostic Laboratory Tests:
 Pus from draining sinuses, sputum, or
specimens of tissue are examined for the
presence of sulfur granules.
The granules are hard, lobulated, and
composed of tissue and bacterial
filaments, which are club-shaped at the
periphery .Specimens are cultured in
thioglycolate broth and on brain-heart
infusion blood
Causing Nocardiosis wich is caused by
infection withNocardia asteroides
complex or, less frequently, Nocardia
brasiliensis or Nocardia otitidiscaviarum,
and only rarely by other species of
nocardia. The Nocardia asteroides complex
includes Nocardia abscessus,
Nocardia farcinia, Nocardia nova,
and others