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Diagnostic microbiology lecture: 12 Gram Positive, Endospore-Forming Bacilli

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Title: Diagnostic microbiology lecture: 12 Gram Positive, Endospore-Forming Bacilli


1
Diagnostic microbiology lecture 12 Gram
Positive, Endospore-Forming Bacilli CLOSTRIDIA A
bed ElKader Elottol MSc. Microbiology 2010
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GENERAL CHARACTERISTICS 1. Anaerobic
gram-positive bacilli 2. Spore formers 3. They
decompose proteins 4. Exotoxin producers 5.
Natural habitat Soil or the intestinal tract of
animals and man. 6. Some motile, others are
non-motile. 7. Catalase negative.
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SPECIES OF MEDICAL IMPORTANCE
  • 1. Clostridium tetani
  • 2. Clostridium perfringens
  • 3. Clostridium botulinum
  • 4. Clostridium septicum
  • 5. Clostridium dificilli
  • 6. Clostridium novyi

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Clostridium tetani
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  • Disease Tetanus or "lock jaw"
  • Cl. tetani is not an invasive organism but rather
    remains strictly localized in the area of the
    affected tissue into which the spores has been
    introduced.
  • The disease is transmitted to man through
    infected wounds.
  • Spores enter into wounds with soil or material
    contaminated with soil.
  • The infection is localized in the area of entry.
  • Growth and toxin production is aided by the
    followings
  • 1. Necrotic tissue
  • 2. Calcium salts
  • 3. Associated pyogenic infection.
  • These conditions aids in providing anaerobic
    conditions.

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  • The toxin "tetanospasmin" is heat-labile protein,
    inactivated by heating for 5 minutes at 65 oC.
  • The toxin may reach the central nervous system.
  • It blocks the release of inhibitory mediators of
    the motor neurons.
  • This results in extreme hyper-reflexia and
    violent spasm of skeletal muscles in response to
    any stimuli.
  • This toxin is inactivated by the proteolytic
    enzymes of the digestive system.
  • 1 mg pure toxin has 6 million lethal mice doses.

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. People have become infected with tetanus
following surgery, burns, abrasions, crush
wounds, ear infections, dental infections, animal
bites, abortion, pregnancy, body piercing and
tattooing, and injection drug use. People can
also get tetanus from splinters.
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The incubation period varies from 3-21 days, most
cases within 14 days with an average of eight
days. The further the injury site is from the
central nervous system, the longer the incubation
period. The shorter the incubation period, the
higher the risk of death
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Neonatal tetanus
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Neonatal tetanus
This baby has neonatal tetanus. It is completely
rigid. Tetanus kills most of the babies who get
it. Infection usually happens when newly cut
umbilical cord is exposed to dirt
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This baby has tetanus. He cannot breast feed or
open his mouth because the muscles in his face
have become so tight
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  • Morphology
  • 1. Gram-positive anaerobic
  • 2. Motile with peritrichous flagella
  • 3.Terminal rounded spores (Drumstick appearance).
  • Media of Choice
  • Any medium with thioglycollate under anaerobic
    condition most especially Cooked Meat Media.

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Tetanus is a life-threatening disease
Treatment consists of two main steps First,
the patient is given antibiotics to kill the
bacteria Metronidazole Or
penicillin Second, injections of antitoxin
are also given. An antitoxin is a substance
that reacts with and destroys the toxin.
Patients may also need medication to control
muscle spasms. In severe cases, the patient
may have to be placed on artificial respiration.
Recovery takes six weeks or more. After a
patient is better, he or she should receive the
tetanus vaccine to protect against future
episodes of the disease.
TREATMENT
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  • vaccination
  • combination of vaccines that protect against
    three diseases, Diphtheria, Tetanus and
    Pertussis DTP vaccine
  • The vaccine is given in five doses at the ages of
    two months, four months, six months,
    fifteen to eighteen months, and four to
    six years.
  • Adults should receive a booster shot against
    tetanus every ten years
  • A booster shot is a dose of the vaccine that
    renews a person's resistance to the disease

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Pregnant also should receive vaccine to
prevent neonatal tetanus for their babies during
cut umbilical cord Make sure that wounds
are thoroughly cleaned. A wound should also be
treated with an antibiotic cream and covered with
a bandage. Wounds that don't heal should be
examined by a doctor
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Clostridium perfringens
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Disease Gas gangrene
  • It causes a variety of infection to man
  • 1. Wound infection
  • 2. Gas gangrene (Myonecrosis)
  • 3. Necrotizing jejunitis
  • 4. Food poisoning
  • 5. Meningitis

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  • Four types of toxins are produced
  • Alpha It is a phospholipase C which hydrolysis
    lecithin.
  • Disrupts cell membranes of RBCs, WBCs muscle
    cells.
  • 2. Beta Is necrotic for the intestinal mucosa
    and lethal to the CNS.
  • 3. Epsilon Inactive which is converted to the
    active form by trypsin in small intestine.
  • pulmonary edema, neurologic symptoms, or
    gastroenteritis could be seen.
  • 4. Iota Similar to epsilon but differ in
    structure

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Subdivided into 5 types based on the four major
lethal toxins they produce. Type A causes most
of the human infections.
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C. perfringens Pathogenesis
Vegetative cell
Spore
Heat
Food
Developing Spore
Free Spore
Toxin inclusion body
Free toxin
GI tract
Cell wall lysis
C. perfringens gastroenteritis can include
diarrhea, nausea, severe abdominal cramps and
bloating for 1-2 days. Vomiting and fever are
not usually seen.  
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Food Poisoning
Spore Contamination
Cooking
Germination
Slow cooling and/or storage at moderate
temperature
Disease cycle
Ingestion
GI illness
Environment
Rapid proliferation
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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.

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  • Morphological Characteristics
  • Spores are usually oval and central or
    subterminal.
  • Not strictly anaerobe and can survive exposure
    to oxygen for short periods of time and sometimes
    referred to as aerotolerant anaerobe.
  • Non-motile
  • They form capsule in infected patients and
    animals.
  • Collagenase, hyaluronidase, and Dnase Enzymes
  • Cultural Characteristics
  • On blood agar plates Colonies are round, domed
    and grayish white, surrounded by a zone of
    hemolysis.

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Micro Macroscopic C. perfringens
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Clostridium botulinum
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  • BOTULISM
  • It is a disease caused by the ingestion of food
    containing the neurotoxin produced by C.
    botulinum.
  • The earliest symptoms usually are an acute
    digestive disturbances followed by nausea and
    vomiting and possibly diarrhea, together with
    fatigue, dizziness and headache.
  • Later there is constipation.
  • Double vision may be evident early and
    difficulty in swallowing and speaking may be
    noted.
  • Patient may complain of dryness of the mouth and
    constriction of the throat.

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  • Involuntary muscles become paralyzed, paralysis
    spread to the respiratory system and heart, the
    death usually results from respiratory failure.
  • INFANT BOTULISM
  • Organisms introduced with dietary supplement
    Organisms multiply in colon with absorption of
    small amount of toxin Infant suffers from
    constipation feeding problems One of the causes
    of sudden infant death.
  • Wound botulism
  • is caused by neurotoxin produced from a wound
    that is infected with the bacteria Clostridium
    botulinum.

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  • Toxicity
  • Botulinum toxin is heat-labile, if heated to a
    certain temperature so can be destroyed if heated
    and held at 80 C for 10 minutes or longer.
  • Botulinum toxin is the most powerful toxin known
    to man (1mg pure toxin has a 20 million lethal
    mice doses).
  • Toxine Classification
  • 8 types has been identified (A-H). Botox
  • Only A, B, E, and F affects human.
  • 1. Type A Most common in western parts of US.
    More toxic than B.
  • 2. Type B Less toxic most frequent in
    occurrence.
  • 3. Type E Obtained from fish and fish products.
  • 4. Type F Similar to A and B. Has been isolated
    in Denmark.

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  • Morphological Characteristics
  • Spores are oval, and subterminal
  • Motile with peritrichous flagella

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Clostridium septicum
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  • This organism sometimes causes rapidly
    progressive cases of Clostridial myonecrosis
  • associated with severe wounds.
  • Apparently, C. septicum, occasionally escapes
    from wounds and is carried by the blood stream to
    other parts of the body.
  • Generalized C.septicum infection may occur in
    leukemia patients or by the administration of
    antimetabolite or other immunosuppressive drugs.

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Clostridium difficile
  • Part of the normal flora of the human gut.
  • It is the major cause of Pseudomembranous
    colitis that sometimes follows the administration
    of broad spectrum antibiotics for prolonged
    periods.
  • Only when the other anaerobes in the intestine
    are killed or inhibited by certain
    antimicrobials, does C. difficile grow in
    sufficient numbers, and produce sufficiently
    great amount of toxin to cause colitis.

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  • This organism produces two toxins
  • Toxin A (an enterotoxin) induces hypersecretion
    of fluid, and development of hemorrhagic
    necrosis.
  • Toxin B (a cytotoxin) causes tissue damage.
  • Disease occurs if the organism proliferates in
    the colon and produces toxins watery or bloody
    diarrhea, abdominal cramps, leukocytosis and
    fever.

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Clostridium novyi (A B)
  • This organism is rarely associated with human
    infection other than clostridial myonecrosis.
  • Type A strains are much more common than type B.
  • Type B strain, almost unknown in human
    infections.
  • They are primarily pathogens of sheep.

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ISOLATION OF CLOSTRIDIA
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  • 1. From Clinical Sample that are Usually Sterile
    (e.g., Blood)
  • Inject the specimen in Blood Culture bottles with
    CO2 and incubate anaerobically in an anaerobic
    jar.
  • Subcultures onto freshly prepared Blood Agar are
    made and incubated anaerobically.
  • 2. From Clinical Specimens that Contain a Mixture
    of Organisms (e.g., Abscess)
  • Heat Treatment
  • Specimen is inoculated to a tube of chopped-meat
    broth medium.
  • Heat at 80 oC for 10 minutes before incubating
    anaerobically.
  • Incubate overnight and subculture to Blood Agar
    and Egg-Yolk Agar.

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  • B. Alcohol Treatment
  • 1 ml of the specimen is placed in a sterile
    screw-capped bottle or tube, add an equal amount
    of 50 ethanol for one hour.
  • Pick up some of the treated materials, and
    inoculate egg yolk agar and blood agar and
    incubate anaerobically.
  • A good selective media is Neomycin-Egg-Yolk
    Agar.
  • NB Some Clostridium species may swarm. To
    prevent swarming, the amount of gar is increased
    to 4-6.
  • NB When growth is evident inoculate the
    following differential media for identification

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  • IDENTIFICATION OF CLOSTRIDIA
  • To differentiate between Clostridia and Bacillus
    especially B. cereus which grow well under
    anaerobic conditions.
  • catalase test is performed which is negative for
    Clostridia and positive for Bacillus.
  • C. perfringens produces lecithinase
    (phospholipase C).
  • Other species produces lipase that break down
    fatty acids.

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  • Identification of toxigenic strains is done in a
    similar way to that of diphtheria by toxin
    neutralization in Vivo.
  • If C. perfringens is to be specifically isolated,
    incubate the plates at 44-46 oC.
  • TREATMENT
  • 1.TETANUS Since treatment of tetanus are not
    satisfactory, prevention is all important.
  • Prevention of tetanus depends upon
  • 1. Active immunization with toxoids
  • 2. Proper care of wounds, contaminated with soil
  • 3. Prophylactic use of antitoxin
  • 4. Administration of penicillin.

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  • 2. BOTULISIM
  • Potent antitoxin to 3 types of botulinus toxins
    have been prepared.
  • Trivalent (A,B,E) antitoxin is administered
    intravenously as early as possible to neutralize
    the effect of the toxin.
  • 3. GAS GANGRENE Treatment of the disease is done
    by
  • 1. Surgical measures to remove all devitalized
    tissues.
  • 2. Administration of penicillin
  • 3. Administration of antitoxin.
  • 4. Infection due to C. septicum and C. novyi
    Similar to gas gangrene
  • 5. Pseudomembraneous colitis Administration of
    oral vancomycin is effective in reducing the
    number of this organism in the human intestine.

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Di glycride formation
Lactose egg yolk milk agar
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END of LECTURE
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