Anaerobic rods causing purulent wound infections. Prevention of Gas gangrene

1. ANAEROBIC RODS CAUSING PURULENT WOUND INFECTIONS.
PATHOGENESIS, LABORATORY DIAGNOSTICS AND SPECIFIC PREVENTION OF
GAS GANGRENE.
I. THEORETICAL QUESTIONS
1. General characteristics of the Clostridium causing gas gangrene.
Taxonomic position.
2. Morphology and cultural characteristics.
3. Biochemical reactions and antigenic structure.
4. Epidemiology and pathogenesis of the gas gangrene.
5. Laboratory diagnostics of the disease.
6. Prophylaxis and treatment of the disease.
Morphology
All of Clostridia are: Gram (+), spore-forming bacilli, about 2-10 x 1-2 μm in size. As a
rule, motile (exception is C.perfringens). The most species are non-capsulated (exception is
C.perfringens). They are pleomorphic in old culteres. May produce long filamentous forms in
human body. During sporulation typical rod-shaped form of cell is changed (the spore is wider
than diameter of bacillus (swollen rod).
Spore may be:
• Central, giving bacillus a spindle shape (C.perfringens)
• Subterminal, the bacillus appearing club shaped (C.perfringens)
• Oval and subterminal, resembling a tennis racket (C.botulinum)
Spherical and terminal, the bacillus is like drumstick (C.tetani).
Cultivation
Clostridia are strictly obligate anaerobic to aerotolerant. The optimum temperature is
370
C, optimum pH is 7-7.4. Clostridia are cultivated on special media for anaerobs as
following:
1. Robertson´s cooked meat broth (the growth appears turbidity, gas production; some
species make meat pink due to sacharolytic features; proteolytic species make the meat
black and produce foul and pervasive odour)
2. Litmus milk media. Clostridia can clot milk with acid an gas production. Acid formation
leads to change the color of litmus from blue to red

2. 3. Kitt-Tarocci´s media is a broth with boiled pieces of liver, kidneys or another
parenchimatic organs, glucose; it is covered liquid sterile vaselin oil to prevent contact
with air. The growth of Clostridia is with turbidity and gas production.
4. Blood sugar agar. Clostridia form smooth or rough colonies surrounded with clear zone
of hemolysis.
Resistance
Vegetative cells of clostridia are high sensitive to heating, oxygen, disinfectants. Under
unfavorable conditions clostridia produce spores which are characterized with high resistance
to heating. Spores survive about 1-4 hours at 1000
C. But all of them are destroyed by
autoclaving at 1210
C within 20 minutes.
Spores can survive in dried soil for several decades. They withstand desinfectants in
ordinary concentration within some hours, can survive into the 700
C ethanol, 1% iodine
solution, 2% phenolic acid solution from 3 hours to 5 days.
Ecology
Clostridia are found worldwide in the soil, in water, in dust, on the plants (ubiquitous
saprophites) as well as in normal intestinal flora of man and animals.Biological role of
clostridia: Intestinal clostridia can invade host ischemic tissues after death and take part in
decomposition cadaver. Virulent factors
The major virulent factors of pathogenic clostridia are exotoxins. They are responsible
for pathogenesis and clinical signs of infections (!). Sometimes enzymes and capsule enhance
virulent abilities of clostridia.
C.perfringens is causative agent of gas gangrene and food poisoning infection
C. hystolyticum additional agents
C.novyi causing
C. septicum gas
C.sporogenes gangrene
C.difficile (also cause pseudomemranous enterocolitis (enteric infection) under specific
conditions. C.perfringens.
Features of morphology: It is large, brick-shaped rod, 4-6x1-2 µm in size, with central or sub-
terminal spore. It is capsulated and non-motile (!).

3. Cultural characteristics Robertson's CMB: It produces gas and a sour odor; the meat is turned
pink. Blood agar: it is cultivated at 450
C (to isolate C.perfringens from mixed culture);
colonies are surrounded with hemolysis. Litmus milk: “stormy fermentation” is proper
sign of C.perfringens (milk is clotted, clot will be disrupted by gas, litmus is turn from blue
to red).
Classification of C.perfringens As some causative agents of gas gangrene C.perfringens is
divided into serotypes based on the range of prodused toxins. Serotype is detected with
toxin-neutralizing test in animals and marked with capitalized letter of Latin alphabet
(A,B,C, D, E).
Virulent factors
• Exotoxins: C.perfringens may form at least 12 distinct exotoxins, which possess hemolytic,
lethal and dermonecrotic properties
• 4 major toxins (α, β, ε, ι) are predominantly responsible for pathogenecity
• Additional toxins (γ,δ, η, κ, λ) have lethal and necrotizing properties
• Enzymes (proteolytic) are collagenase, proteinase, gelatinase, deoxyribonuclease, etc.
• Capsule (antiphagocytic properties)
Pathogenesis.
1. Initial trauma (muscle damage, blood supply impairment and contamination of the soil);
2. sporulation of clostridia;
3. releasing exotoxins
4. Necrotyizing of tissue
5. toxic products of tissue fermentation and toxemia cause severe shock and renal failure
(main reasons of death).
Clinical signs: edematous and discolored muscles in the wound, foul-smelling bubbled
exudates, crepitation of cellular tissue, severe intoxication. Incubation period is about 1-6
days.
Laboratory diagnostics Diagnosis is based on clinical symptoms coupled with :
• Microscopy: there are a large number of Gram-positive bacilli, but no leucocytes in the
smear
• Culture method: it allows to identify causative agents of gas gangrene, detect their
serotypes that important for specific therapy
• Type of exotoxin may be reveal with toxin-neutralazing test in animals

4. Prophylaxis and therapy of gas gangrene
• Surgery prophylaxis is the most important prophylactic measure. All damaged, necrotic
tissues, foreign bodies and blood clots must be removed from the wound
• Passive immunization with anti-gas gangrene serum is rare in use
• Therapy with corresponding antitoxin is necessary to prevent spreading of gas gangrene
• The major point of therapy is adequate local treatment of the wound (antiseptics,
antibiotics, oxygenation).
II.Students practical activities:
a. Microscopy the prepared smears from pure culture of Clostridium stained by
Gram. Estimate the morphology and sketch the image.
b. Familiarize with diagnostic media for cultivation of anaerobs.
c. Detect the biochemical features of Clostridium accoding to growth results
into the Hiss media. Note them in protocol.
4. Write down the principal scheme of laboratory diagnostics of the gas gangrene.