ANAEROBIC

SPORE-FORMING
BACTERIA
S.Y.Maselle

Spore-forming anaerobes
These are bacilli
They are Gram positive.
They are generally oxygen intolerant
They form spores
They belong to ONE Genus: CLOSTRIDIUM.

CLOSTRIDIUM
Gram positive spore-forming anaerobic bacilli.
Most of them are free-living saprophytes

occurring in soil, water and decomposing

plant and animal matter.
Some such as Clostridium perfringens are
commensals of the animal and human gastrointestinal tract.
A few species are opportunistic pathogens.

General description of the Genus

Large straight or curved rods with rounded

ends.
Pleumorphism is common eg elongated,
filaments, spindles and club forms.
Gram positive but may be Gram variable.
All form spores which enable the organisms to
survive in adverse conditions
Most of them are obligate anaerobes

General description
cont..
A few species can tolerate small amounts of

oxygen eg Clostridium perfringens.

They are biochemically active either with
saccharolytic or proteolytic or both properties.
Many species are highly toxigenic
The toxins may be neurotoxins, histotoxins
or enterotoxins.

Clostridium species
Gas gangrene group-

C.perfringens
C.septicum
C.novyi ( oedimatiens)
C.sordelli
C.histolyticum
C.tetani
C.botulinum
C.difficile
Non-pathogenic: C.bifermentans, C.sporogenes, C
tertium.
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Clostridium perfringens
(C.welchii)
It is the prototype species of the genus Clostridium
Commonly implicated in gas gangrene and food

poisoning.
Strongly Gram positive rods brick shaped with blunt
ends.
Capsulated if freshly isolated and non-motile
Grows quickly in the lab especially at high
temperatures,~ 42oC where doubling time will be about 8
minutes.
Can tolerate small amounts of oxygen
Does NOT sporulate readily: needs special medium
to sporulate in the lab ( Ellners medium)
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C PERFRINGENS....
Typed on the basis of the major lethal toxins
it produses which are alpha (), beta (),epsilon ()
and iota () as shown in the table below
The five types of C perfringens are A to E
Type A is responsible for the classical gas

gangrene
A sutype of type A responsible for food
poisoning
Type C causes pig bel
The other types cause disease in animals.
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Distribution of major lethal toxins of

C perfringens Types A to E.
Type
Iota
A
B
C
D

Major
Alpha

lethal
Beta

toxins
epsilon

Cultural characteristics of
Clostridium prefringens
Will grow under anaerobic conditions but can

grow in presence of small amounts of oxygen

(microaerophilic)
Grows well on blood agar medium
After 24 hrs incubation produces large
colonies 2-3 mm in diameter , round, smooth,
regular, convex, with Beta haemolysis.
On horse-blood, colonies of C perfrngens
type A are surrounded by a small zone of
complete haemolysis and a wider zone of
incomplete haemolysis, a phenomenon known
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C.perfringens:
Identification
Strongly gram positive rods , short, regular with

rounded ends.
Spores rarely seen in vivo or in vitro.
Non-motile
Capsulated if freshly isolated
Colonial morphology on blood agar producing
target-haemolysis due to and haemolysins
produced by C.perfringens ( produces small
zone of complete haemolysis surrounded by a
wider zone of incomplete haemolysis caused by
-toxin).
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NOTE: Large
rectangular
grampositive bacilli

NOTE: Double zone of

hemolysis

Inner beta-hemolysis =

toxin Outer alphahemolysis = toxin

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C.perfringens:
Identification
Stormy clot in litmus milk due to acid and gas

production from fermentation of lactose in milk

Nagler reaction- due to action of its phospholipase on
egg yolk medium where colonies are surrounded by
zones of opacity and this effect is specifically inhibited
by C.perfringens antiserum containing -antitoxin
Egg yolk is rich in lecithin and the opacity is due to
production of insoluble proteins and fatty acids due to
phospholipase,(lecithinase C).
Lecithinase C is the -toxin responsible for haemolysis on BA.
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Nagler reaction

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Gas gangrene
C.perfringens is the commonest cause of gas

gangrene.
Other species eg C.septicum,
C.novyi,C.sordelli and C.histolyticum may be
responsible too.
Disease characterized by rapidly spreading
edema, myositis, tissue necrosis and gas in
the tissues with profound toxaemia.
Often follows wound infection with
C.perfringens
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Clostridial cellulitis

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Gas gangrene
Main source of the organism is animal or

human excreta. Spores of the organism are

widely distributed in nature.
They are present in soil, street dust, air or
dirty clothing.
Normal skin may also bear spores of
Clostridium especially in areas contaminated
with intestinal organisms like the perineum,
buttocks and the lower limbs.

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Gas gangrene
Impairment of blood supply, massive tissue

injury with severing of blood supply, presence of

dead tissues and foreign body are among the
factors which promote development of gas
gangrene in the wound.
Debility, old age and diabetes are predisposing
factors .
Gas gangrene of the uterus may arise from
septic criminal abortion.
Diagnosis of gas gangrene is essentially clinical.
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Gas gangrene
However laboratory confirmation is by

isolation of the organism.

Treatment is by surgical removal of all dead
tissue and foreign bodies, and administration
of antibiotics.
Amputation of affected limbs or organs is
often necessary.
Hyperbaric oxygen if available may be helpful.
No vaccines so far.
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C.perfringens food
poisoning
Due to strain of C perfringens Type A which

produces spores which are highly resistant to heat.

Meat is often contaminated with such spores
Heat resistant spores will survive cooking and
germinate during cooling.
Consumption of such meat will result in food
poisoning mediated by an enterotoxin.
The enterotoxin is produced in the large intestine
when the bacteria sporulate.
Treatment is symptomatic and prevention is by
good food hygiene.
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CLOSTRIDIUM TETANI
On microscopy appears as straight slender

Gram positive rod with terminal round spores

which give the organism the appearance of a
drumstick.
Obligate anaerobe and it is motile
When grown on BA will produce a thin
spreading film swarmming growth with
-haemolysis
Spores highly resistant to heat and
disinfectants.
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C tetani spores

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C.tetani toxins.
Oxygen labile& heat stable haemolysin

( tetanolysin) but it is of unknown clinical

significance.
Neurotoxin ( tetanospasmin) which is heatlabile is responsible for the disease tetanus.
The gene that encodes the neurotoxin is
located on 75-kb plasmid which is nonconjugative, thus a nontoxic strain cannot be
converted to a toxigenic strain.
The toxin is toxic to man and other animals.
The lethal dose for a mouse is 0.0001g.
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Clinical tetanus
Following wound infection tetanus bacilli are not

invasive, they remain and multiply at initial site.

Toxin is produced and diffuses to relevant level of
spinal cord and the entire CNS
Once the toxin is internalised into the presynaptic
cells it prevents the release of the inhibitory
neuro-transmitters glycerine and -aminobutyric
acid.
Motor neurons are left without inhibitory control,
undergo sustained excitation resulting in the
characteristic motor spasms of tetanus.
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Clinical tetanus
Onset of symptoms is gradual often starting with

stiffness of the jaw ( lockjaw).

Pain and stiffness of neck and back follows.
Stiffness spreads to all muscle groups which may
result in sardonic grin or resus sardonicus
and spasms of back muscles results in
opisthotonos.
Generalized spasms is elicited by any external
stimuli eg light, noise , touch etc.
Sweating, tachycardia and swings of BP and body
temperature.
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Clinical tetanus
Supportive treatment which include

maintaining breathing, hydration and feeding.

Avoid any stimuli which could elicit spasms.
Isolate in a dark quiet room
Administration of antitoxin ,sedation and
other supportive nursing care.
If wound obvious, surgical toilet and penicillin
or metronidazole given to stop bacterial
growth and toxin production.
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Tetanus neonatorum
Neonatal tetanus is typically associated with

an initial infection of the umbilical stump.

This is often due to the use of unsterile
instruments to cut the umbilical cord
Also dressing the umbilical stump with
unsterile dressings or use cow dung to
facilitate healing of umbilical sump.
This is almost exclusively a disease in
developing countries and mortality in infants
exceeds 90%.
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Neonatal tetanus

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Neonatal tetanus

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Prevention and control of tetanus.

Prompt and adequate wound toilet essential.
Prophylaxis with tetanus antiserum ( human) for non-

immune patients plus tetanus vaccine full course of

three doses of tetanus toxoid (TT).
For immune patients only tetanus toxoid booster dose.
Vaccination of women in pregnancy prevents tetanus
neonatorum. This is also the practice in Tanzania.
Vaccination of children as part of Imminisation and
Vaccine Development Programme IVDP ( EPI) has
drastically reduced tetanus in many countries.

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CLOSTRIDIUM
BOTULINUM
Strict anaerobe, motile with oval subterminal spores
Widely distributed in natue
Produces potent neurotoxin in the food
There are seven types A-G but types A,B and E are

most common.
Botulism is a severe and often fatal disease
Foods affected mostly preserved meats, vegetables,
canned fish and honey
Preformed toxin in food is absorbed and binds to
presynaptic nerve endings where it inhibits
acetylcholine.
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CLOSTRIDIUM
BOTULINUM
Inhibition of acetlycholine blocks

neurotransmission at the peripheral

cholinergic synapses.
Since acetylcholine is required for excitation
of muscle, the resulting clinical presentation
of botulism is a flaccid paralysis.
As with tetanus, recovery of function requires
regeneration of the nerve endings.

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CLOSTRIDIUM
BOTULINUM
Incubation period 1-2 days
Oculomotor muscles affected initially, then

spreads to involve other muscles.

Initial symptoms may be diplopia and squint
Progressive flaccid paralysis
Death due paralysis of muscles of respiration.
Treatment involves giving antitoxin and
intesive nursing care
Vaccine availabe.
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CLOSTRIDIUM DIFFICILE
Until mid-1970s the clinical importance of C difficile was

not known.
It is now known to be associated with human disease.
Gram positive,motile,subterminal oval spores and a
strict anaerobe
Colonises about 5% of healthy people and hospitalized
patients.
The organism is relatively resistant to antibiotics
Exposure to antibiotics leads to overgrowth of C difficile
in the colon and subsequent disease development.
Spores can be detected in hospital rooms of infected
patients, hence source of nosocomial infection.
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CLOSTRIDIUM DIFFICILE
Produces toxin A , an enterotoxin and toxin B,a

cytotoxin which probably contribute to pathogenesis of

disease.
Associated with post antibiotic diarrhoea which may
lead to a serious condition called pseudomembranous
colitis.
Diagnosis by isolation of the organism or by detection
of the toxins in faeces using immunoassays.
Discontinuation of the implicated antibiotic is essential
to alleviate the symptoms.
Vancomycin or metronidazole may be given if severe
Prevention is by appropriate antibiotic use.
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LAST SLIDE

GOOD

MORNING.
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