2. Genus Clostridium
• In Anaerobic spore
bearing Gram positive
bacilli Spores are wider
than the body giving
spindle shape
• The name derived
from word Kolster
meaning spindle
Dr.T.V.Rao MD 2
3. Clostridium
C. perfringens: gas gangrene; food poisoning
C. tetani: tetanus
C. botulinum: botulism
C. difficile: pseudomembranous colitis
Physiology and Structure
Anaerobic.
Large gram-positive rods.
The spores are usually wider
than the rods, and are located
terminally or sub terminally.
Most clostridia are motile by
peritrichous flagella.
Dr.T.V.Rao MD 3
4. Clostridia
• Large Gram positive bacilli.
• Straight or slightly curved rods with
slightly rounded ends
• Anaerobic bacilli
• Spore bearing
• Spore do not germinate and growth does
not normally proceed unless a suitably
low redox potential Eh exists
• Many are Saprophytes
Dr.T.V.Rao MD 4
5. SHAPES OF CLOSTIDIA
The shape an position of spores varies in different
species and is useful the identification of Clostridia
Central and equatorial in Cl.bifermentans
Sub terminal in C.perfringens
Oval or terminal in Cl.tertium
Spherical and terminal giving drum stick in C.tetani
Dr.T.V.Rao MD 5
6. Some are Commensals But
Can be Pathogenic
• Some are commensals of the animal &
human gut which invade the blood and
tissue when host die and initiate the
decomposition of the corpse (dead body)
• Causes diseases such as gas gangrene,
tetanus, botulism & pseudo-
membranous colitis by producing toxins
which attack the neurons pathways
Dr.T.V.Rao MD 6
7. Clostridia
• Clostridia are biochemically active,
frequently possessing both saccharolytic
and proteolytic properties, although in
varying degrees. Many species are highly
toxigenic. The toxins produced by the
organisms of tetanus and botulism attack
nervous pathways and are referred to as
neurotoxins.
Dr.T.V.Rao MD 7
8. Clostridia of medical importance
Clostridium
Causing
Antibiotic
Tetanus Botulism
Gas gangrene associated diarrhea
e.g. Cl. tetani e.g. Cl. botulinum
e.g. Cl. difficille
Saccharolytic
Proteolytic Mixed: Cl.
e.g. Cl. perfringens
e.g. Cl. sporogenes histolyticum
&Cl. septicum
Dr.T.V.Rao MD 8
9. How they appear in Gram staining
• They are Gram
positive, but may
appear to be Gram
negative. All produce
spores, which enable
the organisms to
survive in adverse
conditions, for
example in soil and
dust and on skin. Dr.T.V.Rao MD 9
10. Clostridium perfringens
• Large Gram-positive bacilli with stubby
ends
• Capsulated
• Non motile Anaerobic
• Grown quickly on selective media
• Can be identified by Nagler reaction
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11. Some Clostridia Produce Gas
gangrene
• The organisms
associated with gas
gangrene attack soft
tissues by producing
toxins and aggressins,
and are referred to as
histotoxic. C. difficile
and some strains of
C. perfringens
produce enterotoxins.
Dr.T.V.Rao MD 11
12. C. perfringens
• C. perfringens is a
relatively large Gram-
positive bacillus (about 4-
6 × 1 μm) with blunt
ends. It is capsulate and
non-motile. It grows
quickly on laboratory
media, particularly at high
temperatures
(approximately 42°C),
when the doubling time
can be as short as 8 min. I
Dr.T.V.Rao MD 12
13. Clostridium Causing Gas Gangrene
Clostridia causing gas
gangrene
Proteolytic organisms
Saccharolytic organisms
Cl. sporogenes Mixed saccharolytic &
Cl. perfringens, Cl. septicum
Digest proteins with proteolytic
Ferment carbohydrates
blackening bad smell Cl. histolyticum
Acid and gas are produced
production
Dr.T.V.Rao MD 13
14. The Agent
• Clostridium perfringens
– Gram-positive bacteria
• Anaerobic rod
• 3-8 x 0.4 – 1.2 mu
– Found in soil, decaying matter and intestinal
tract of mammals
• 5 types (A-E)
– Types B and D produce the epsilon toxin
Dr.T.V.Rao MD 14
15. Micro & Macroscopic C. perfringens
NOTE: Large rectangular NOTE: Double zone of hemolysis
gram-positive bacilli
Inner beta-hemolysis = θ toxin
Outer alpha-hemolysis = α toxin
Dr.T.V.Rao MD 15
16. Resistance
• Vegetative bacteria is killed like other bacteria
• Cl.perfringens destroyed by boiling
• Cl, botulinum not killed even at 105 c 0 for
less than 100 minutes
• All spores are killed at 121 oc in 20 minutes
• Halogens , Glutaraldehyde are effective on
spores
• Metronidazole and Pencillin and
Chloramphenicol are effective
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17. How Clostridia are Cultivated
• Clostridia grow
well on
ordinary
medium under
anaerobic
medium
Dr.T.V.Rao MD 17
18. Media used for Cultivation
• Liquid medium for
cultivation cooked meat
broth
• Thiglyclolate broth
• CMB contain unsaturated
fatty acids which take up
oxygen
• Proteolytic medium turns
the medium black and
Saccharolytic medium
turn the meat pink
Dr.T.V.Rao MD 18
20. Toxins
• The toxins of Cl. perfringens
– toxin (phospholipase C, lecithinase) is the most
important toxin
• Lyses of RBCs, platelets, leucocytes and endothelial cells
• Increased vascular permeability with massive hemolysis and
bleeding tissue destruction
• Hepatic toxicity and myocardial dysfunction
– -toxin is responsible for necrotic lesions in necrotizing
enterocolitis
– Enterotoxin is heat labile toxin produced in colon →
food poisoning
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21. Epsilon Toxin
• Produced as an inactive protoxin
• Activated by trypsin
– Removes a 13-residue
N-terminal peptide
• Increases intestinal permeability
• Increases vascular permeability
– Vascular damage and edema in brain, heart, lung
and kidneys
Dr.T.V.Rao MD 21
22. Pathology
• Not highly invasive; requires damaged
and dead tissue and anaerobic
conditions
• Conditions stimulate spore
germination, vegetative growth and
release of exotoxins, and other
virulence factors.
• Fermentation of muscle carbohydrates
results in the formation of gas and
further destruction of tissue.
Dr.T.V.Rao MD 22
23. Clinical Diseases
C. perfringens
Gas gangrene
Spores germinate vegetative cells multiply, ferment carbohydrates
and produce gas in the tissue. This results in distension of tissue and
interference with blood supply the bacteria produce necrotizing toxin
and Hyaluronidase, which favor the spread of infection tissue
necrosis extends, resulting in increased bacterial growth, hemolytic
anemia, then severe toxemia and death.
Incubation: 1-7 days after infection.
Symptoms: Crepitation in the subcutaneous tissue and muscle, foul
smelling discharge, rapidly progressing necrosis, fever, hemolysis,
toxemia, shock, renal failure, and death.
Can be also caused by other Clostridium species.
Dr.T.V.Rao MD 23
24. Mechanism of Clostridial
Infections
• When Clostridial infection has been initiated
in a focus of devitalized anaerobic tissue, the
organisms multiply rapidly and produce a
range of toxins and aggressins. These damage
tissue by various necrotizing effects, and some
have demonstrable lethal effects. They spread
into adjacent viable tissue, particularly
muscle, kill it, and render it anaerobic and
vulnerable to further colonization, with the
production of more toxins and aggressins.
Dr.T.V.Rao MD 24
25. Toxic and Enzymatic Mechanisms
Hyaluronidase produced by C. perfringens breaks
down intercellular cement substance and promotes
the spread of the infection along tissue planes.
Collagenase and other proteinases break down
tissues and virtually liquefy muscles. The whole of a
muscle group or segment of a limb may be affected.
α-Toxin, a phospholipase C (lecithinase), is generally
considered to be the main cause of the toxaemia
associated with gas gangrene, although other
Clostridial species can produce similar manifestations.
Dr.T.V.Rao MD 25
28. C. perfringens
Clinical Diseases
Food poisoning
The enterotoxin causes marked hyper secretion in jejunum
and ileum.
Enterotoxin: a heat-labile protein produced by some strains of
C. perfringens type A. When >108 cells in contaminated meat
are ingested and sporulate in the small intestine, enterotoxin is
formed. It disrupts ion transport in the enterocytes, and
induces antibodies (non-protective) in adults.
Symptoms: diarrhea, usually without vomiting or fever.
Necrotizing enteritis (pig-bel): a fatal disease (acute necrosis
in jejunum attributed to -toxin) in children in New Guinea
caused by type C C. perfringens.
Clostridium bacteremia usually occurs in patients with tumors.
Dr.T.V.Rao MD 28
29. Laboratory Diagnosis
Specimen: Histological specimen or wound exudates
Histological specimen transferred aseptically into a sterile screw-capped
bottle & used immediately for microscopical examination & culture
Specimens of exudates should be taken from the deeper areas of the
wound where the infection seems to be most pronounced
Microscopical examination (Gram, Spore stain etc)
Gram-positive bacilli, non motile, capsulated & sporulated
The spore is oval, sub-terminal & non bulging
Spores are rarely observed
Culture: Anaerobically at 37C
On Robertson's cooked meat medium → blackening of meat will observed
with the production of H2S and NH3
On blood agar → β-hemolytic colonies
Dr.T.V.Rao MD 29
32. C. perfringens
Laboratory Diagnosis
Specimens: pus, necrotic tissue, feces, food, etc.
Smears: large gram-positive rods with or without spores, usually in the
absence of leukocytes.
Culture: anaerobic culture on blood plate.
Identification:
“Storming fermentation”-- clot
torn by gas in 24 hrs.
Lecithinase test-- precipitate
formed around colonies on egg
yolk media.
Biochemical tests.
Dr.T.V.Rao MD 32
33. Nagler’s Reaction
• This test is done to detect the lecithinase activity
– The M.O is inoculated on the medium containing
human serum or 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
Dr.T.V.Rao MD 33
34. 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.34
Dr.T.V.Rao MD
36. Collecting a Infected Tissues
• If there are sloughs 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. Gram smears are prepared.
Dr.T.V.Rao MD 36
37. Proceeding with our observations
• If gas gangrene exists, typical Gram-
positive bacilli may predominate, often
with other bacteria present in a mixed
infection. However, there is usually a
pronounced lack of inflammatory cells.
Initiation of treatment should not await
a full laboratory report and early
discussion with the bacteriologist is
crucial.
Dr.T.V.Rao MD 37
38. Importance of Direct Smears
• A direct smear of a wound exudate is
often of great help in providing evidence
of the relative numbers of different
bacteria that may be participating in a
mixed infection or may merely be
present as contaminants, but the
distinction is not invariably easy and joint
discussions are important.
Dr.T.V.Rao MD 38
39. Biochemical Tests
• Cl. perfringnes
characterized by:
It ferments many
carbohydrates with acid
& gas
It acidified litmus milk
with stormy clot
production
Nagler reaction is
positive
Dr.T.V.Rao MD 39
40. Treatment
C. perfringens
Treatment for Suppurative myositis or myonecrosis:
Prompt and extensive debridement.
Antibiotics (penicillin) administration.
Hyperbaric oxygen may "detoxify" patients rapidly.
Efficacy of antitoxins is doubtful.
C. perfringens food poisoning requires only symptomatic care.
Prevention, and Control
Preventive measures: surgical debridement and prophylactic
antibiotics.
Dr.T.V.Rao MD 40
41. Treatment and Prevention
• Immediate cleansing of dirty wounds,
deep wounds, decubitus ulcers,
compound fractures, and infected
incisions
• Debridement of disease tissue
• Large doses of cephalosporin or penicillin
• Hyperbaric oxygen therapy
• No vaccines available
Dr.T.V.Rao MD 41
42. Importance of Prompt Antibiotic
Treatment
• Antibiotic therapy is started immediately in very
high doses. This must take account of the likely
coexistence of coliform organisms, Gram-positive
cocci and faecal anaerobes. Accordingly,
penicillin, metronidazole and an aminoglycoside
may be given in combination. Alternatively,
clindamycin plus an aminoglycoside or a broad-
spectrum antibiotic, such as meropenem or
imipenem, may be considered. Much intensive
supportive therapy is needed.
Dr.T.V.Rao MD 42
43. Surgical attention a Urgent priority
• Prompt and adequate surgical attention to the
wound is of the utmost importance
• Sutures are removed, and necrotic and devitalized
tissue is excised with careful debridement.
• Facial compartments are incised to release tension.
• Any foreign body is found and removed.
• The wound is not resutured but is left open after
thorough cleansing, and loosely packed.
Dr.T.V.Rao MD 43
44. Use of Anti Gas gangrene High
titre serum ?
• The experts have
opined that use of Anti
Gas Gangrene Serum
injection is no longer
the preferred treatment
for such patients. For
the stated reasons, this
drug is not in use and
opinions may differ
Dr.T.V.Rao MD 44
45. Food poisoning strains' of
C. perfringens
• Few strains of C. perfringens can cause food
posinoing.Carrier rates for 'typical food
poisoning strains' of C. perfringens range from
about 2% to more than 30% in different
surveys across the world. These bacteria also
occur in animals; thus, meat is often
contaminated with heat-resistant spores.
When meat is cooked in bulk, heat
penetration and subsequent cooling is slow
unless special cooking precautions are taken.
Dr.T.V.Rao MD 45
46. The pathogens propagate in spite
of cooking
• During the cooling period surviving
spores may germinate and multiply
in the anaerobic environment
produced by the cooked meat.
Anyone eating this will consume the
equivalent of a cooked meat broth
culture of the organism.
Dr.T.V.Rao MD 46
47. The organisms are protected
• The organisms are protected from the
gastric acid by the protein in the meal
and pass in large numbers into the
intestine.
• The vehicle of infection is usually a pre-
cooked meat food that has been allowed
to stand at a temperature conducive to
the multiplication of C. perfringens
Dr.T.V.Rao MD 47
48. ENTERITIS NECROTICANS (PIGBEL)
Subgroup of C. perfringens type C that produces
heat-resistant spores is the cause of a disease
that affects New Guinea natives when they have
pork feasts. The method of cooking the pork
allows the clostridia to survive. When the
contaminated meat is eaten along with a sweet
potato vegetable that contains a proteinase
inhibitor, a toxin (the β-toxin) is able to act on
the small intestine to produce a necrotizing
enteritis. A successful vaccination programme
has reduced the incidence of pigbel dramatically
Dr.T.V.Rao MD 48
49. Diagnosis of Food poising Strains
• Diagnosis depends upon the isolation
of similar strains of C. perfringens from
the faeces of patients and from others
at risk who have eaten the suspected
food, and from the food itself. Numbers
usually exceed 106 organisms/g faeces.
The isolates can be sent to a reference
laboratory for special typing to prove
their relatedness.
Dr.T.V.Rao MD 49
50. Care of the Food a Priority
• The occurrence of this type of food
poisoning is an indictment of the
catering practices concerned, as food
has to be mishandled to allow the
chain of events to take place.
Nevertheless,
How ever C. perfringens is among the
most common causes of food poisoning.
Dr.T.V.Rao MD 50
51. • Programme Created by Dr.T.V.Rao MD
for Medical and Paramedical Students in
Developing World
• Email
• doctortvrao@gmail.com
Dr.T.V.Rao MD 51
