Clostridium species - Microbiology

College Of Dentistry - Mosul University

Clostridium
Gram-positive . obligate
anaerobes capable of
producing endospores which
protect them in harmful
environment . Individual cells
are rod shaped.
The spores are usually wider
than the rods, and are located
terminally or sub terminally.
Most clostridia are motile by
peritrichous flagella.while
others have a capsule like
Clostridium.perfringens
Characteristics
College Of Dentistry - Mosul University 2

Clostridium consists of around 100 species that
include common free-living bacteria as well as
important pathogens There are five main
species
responsible for disease in
humans.
C. perfringens: gas gangrene; food poisoning
C. tetani: tetanus
C. botulinum: botulism
C. difficile: pseudomembranous colitis
C.Sordellii : can cause a fatal infection in exceptionally rare
cases after medical abortions

The shape an position of spores varies in different
species and is useful the identification of
Clostridia
*Central in Cl.bifermentans
*Sub terminal in Cl.perfringens
*Oval or terminal in Cl.tertium
*Spherical and terminal giving
drum stick appearance in Cl.tetani

 C. perfringens is a
relatively large Gram-
positive short fat
bacilli with blunt ends.
It is capsulate and
non-motile. Anaerobic.
It grows quickly on
laboratory media on
blood agar ( B –
Haemolytic )

 Liquid medium for
cultivation cooked
meat broth
 Thiglyclolate broth
 CMB contain
unsaturated fatty
acids which take up
oxygen

Clostridium. perfringens
Culture & identification

It distinguish between different
species of bacteria.
The lactose (milk
sugar), litmus (pH indicator),
and casein(milk protein)
contained within the medium can
all be metabolized by different
types of bacteria.
Milk is the first substrate used to
maintain bacteria, this test allows
for accurate depiction of bacterial
types. The addition of litmus,
other than explaining the pH type,
acts as an oxidation-reduction
indicator. The test itself tells
whether the bacterium can
ferment lactose, reduce litmus,
form clots, form gas, College Of Dentistry - Mosul University

 This test is done to detect the lecithinase
activity
 The M.O is streaked on the medium containing egg
yolk (contains lecithin)
 The plate is incubated anaerobically at 37 C for 24 h
 Colonies of Cl. perfringens are surrounded by zones
of turbidity due to lecithinase activity and the effect is
specifically inhibited if Cl. perfringens antiserum
containing antitoxin is present on the medium
 `

C. perfringens Nagler Reaction
NOTE: Lecithinase (α-toxin; phospholipase) hydrolyzes
phospholipids in egg-yolk agar around streak on right.
Antibody against α-toxin inhibits activity around left streak.

Positive Nagler Reaction
Procedure of Nagler Reaction

Anaerobic Jar

 The organisms
associated with gas
gangrene attack soft
tissues by producing
toxins and aggressins,
and some strains of
the bacteria produce
enterotoxins and
cause food poisoning

 If there are pieces of necrotic tissue in
the wound, small pieces should be
transferred aseptically into a sterile
screw-capped bottle and examined
immediately by microscopy and culture.
Specimens of exudate should be taken
from the deeper areas of the wound
where the infection seems to be most
pronounced.

 Virulence factors
 toxins –
 alpha toxin – causes RBC rupture,
edema and tissue destruction
 Enterotoxin
 collagenase
 Hyaluronidase
 DNase

Clostridium tetani
Anaerobic bacteria of the genus species Clostridium it is gram
positive, slender bacillus and it has spherical terminal spores
giving drum stick appearance
It is non capsulated & motile with peritrichus flagella
It produces a potent biological toxin, tetanospasmin, and is the
causative agent of tetanus a disease characterized by painful
muscular spasms that can lead to respiratory failure and, in up
to 40% of cases, death.

 An infectious disease caused by
contamination of wounds from the
bacteria Clostridium tetani, or the
spores they produce that live in the soil,
and animal feces
 Infection follows when spores
 become activated and develop
 into gram-positive bacteria that multiply
 and produce a very powerful toxin
(tetanospasmin) that affects the
muscles.

 Tetanus spores are found throughout the
environment, usually in soil, dust, and animal waste.
 Tetanus is acquired through contact with the
environment; it is not transmitted from person to
person.

 The usual locations for the bacteria to enter the
body:
 Puncture wounds (such as those caused by rusty
nails, splinters, or insect bites.)
 Burns, any break in the skin, and IV drug access sites are
also potential entryways for the bacteria.

1. It inhibits the release of acetylcholine thus it
interferes with neuromuscular transmission.

2. Inhibition of postsynaptic spinal neurons by
blocking the release of an inhibiting mediator

 Gram +ve stains grow on blood agar media
aerobically
 Inoculation of culture in to 2 mice one is protected
with anti-toxin and the other is unprotected (dies with
typical tetanic spasms )

"C. diff", is a species
positive-Gramof
of the genusbacteria
thatClostridium
causes diarrhea and
other intestinal
disease when
competing bacteria
are wiped out by
antibiotics.

Most common cause of nosocomial diarrhea.
Rate and severity of C. difficile-associated diarrhea
(CDAD) increasing.
Clostridium difficile is a bacterium that can cause
symptoms ranging from diarrhea to life-threatening
inflammation of the colon. Illness from C. difficile
most commonly affects older adults in hospitals or
in long term care facilities and typically occurs after
use of antibiotic medication

characteristic
features:
Clostridia are
anaerobic, spore-
forming rods (bacilli).

C. difficile bacteria can be found throughout the
environment — in soil, air, water, and human
and animal feces. A small number of healthy
people naturally carry the bacteria in their large
intestine. But C. difficile is most common in
hospitals and other health care facilities, where
a much higher percentage of people carry the
bacteria.

Reservoir
Infectious Agent
C.difficile
Means of
Transmission
Portal of entry
Susceptible Host
Chainof infection
Bowel and
Contaminated
environment
Contact
transmission from
contaminated
hands,
equipment or the
environment
Faecal/Oral
>65 years
History of antibiotic use
Recent received
healthcare
Underlying conditions
Abdominal surgery
Weakened immunity

Disruption of normal
colonic flora
Colonisation with C.
difficile
Production of toxin A
+/- B
Mucosal injury and
inflammation

Toxigenic strains
produce 2 major
toxins:
toxin A
(enterotoxin)
toxin B (cytotoxin)
Neutralised by C.
sordellii antitoxin

Watery diarrhea three or more times a day for two
or more days
Mild abdominal cramping and tenderness
Watery diarrhea 10 to 15 times a day
Abdominal cramping and pain, which may be
severe
Fever
Blood or pus in the stool
Nausea
Dehydration
Loss of appetite
Weight loss

Pseudomembraneous colitis
Perforation of the colon
Sepsis
Death

The Specimen
Fresh is best (test within 2 hours)
Liquid or loose, not solid
If unable to test within 2 hours, refrigerate
at 4 C for up to 3 days
Freeze at -70 C (not -20 C) if testing will
be delayed
Specimen quality will influence test results

Endoscopy
(pseudomembranous
colitis)
Culture
Cell culture cytotoxin
test
EIA toxin test
PCR toxin gene
detection

Note : alcohol does not kill C. difficile spores,
use of soap and water is more efficacious

Gram positive
Obligate anaerobic bacillus
Spores
Resistant to heat, light, drying and radiation
Specific conditions for germination
Anaerobic conditions
Warmth (10-50oC)
Mild alkalinity

Ingestion
Organism
Spores
Neurotoxin
Wound contamination
Inhalation
Person-to-person not documented

Three forms
Foodborne
Wound
Infant
All forms fatal and a medical emergency
Incubation period: 12-36 hours

The symptoms of botulism are similar to
those of Guillain-Barré syndrome, stroke,
and myasthenia gravis.
As a result, botulism is probably
substantially under-diagnosed.
Serum electrolytes, renal and liver function
tests, complete blood tests, urinalysis, and
electrocardiograms will all be normal unless
secondary complications occur.

The incubation period varies according to
the mode of transmission, rate of absorption
of the toxin, and the total amount and type
of toxin.
Foodborne botulism usually takes 24-36
hours to manifest itself.
Wound botulism often takes 3 or more days
to appear.
Inhalation botulism has occurred very rarely,
but incubation times may range from several
hours to perhaps days, again depending
upon the type and amount of toxin inhaled.

All four types of botulism result in symmetric
descending flaccid paralysis of motor and
autonomic nerves always beginning with the
cranial nerves. These symptoms are
preceded by constipation in cases of infant
botulism.
Symptoms include:
Double or blurred vision
Drooping eyelids
Dry mouth
Difficulty Swallowing
Muscle weakness

If left untreated symptoms may expand to
include paralysis of respiratory muscles as
well as the arms and legs.
Asphyxiation due to respiratory paralysis is
the most common cause of death in
botulism cases.

Proper food preparation is one of the most
effective ways to limit the risk of exposure to
botulism toxin.
Boiling food or water for ten minutes can
eliminate some strains of Clostridium botulinum
as well as neutralize the toxin as well.
However, this will not assure 100% elimination.
Limiting growth of Clostridium botulinum and
the production of botulism toxin is an
alternative to their outright destruction.

Now manufactured under the name ―Botox‖
Experimentally used for treating migraine
headaches, chronic low back pain, stroke,
cerebral palsy, and dystonias (neurologic
diseases involving abnormal muscle posture
and tension)
Frequent injections allows an individual to
develop antibodies
Studies carried out to determine feasibility of
other strains of BoNT
BoNT B manufactured for treatment of
cervical dystonia in 2000 as ―Myobloc‖

Botox injection patient 13 weeks after injection
Sadick, N. and A.R. Herman (2003). “Comparison of Botulinum Toxins A and B in the
Aesthetic Treatment of Facial Rhytides.” Dermatologic Surgery 29:340-347.

Clostridium species - Microbiology

Clostridium species - Microbiology

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