The CLOSTRIDIUM SPECIES

By.
Assist. Prof.Dr. Nada H. Al-Mudallal

This Lecture contains the following objects:

• Clostridium species.
• Morphology and Identification.
• Culture.
• Colony form.
• Growth characteristics.
• Antigenic characteristics.
• C. Botulinium
• C.tetani.
• C. Perfringes
• C. difficile.
• Toxins
• Pathogenesis.
• Clinical finding.
• Diagnostic Laboratory test.
• Treatment
 CLOSTRIDIUM SPECIES
 The clostridia are large anaerobic, gram-positive, motile rods.
 Many decompose proteins or form toxins, and some do both.
 Their natural habitat is the soil or the intestinal tract of animals and
humans, where they live as saprophytes. Among the pathogens are the
organisms causing botulism, tetanus, gas gangrene, and
pseudomembranous colitis.
Morphology & Identification
TYPICAL ORGANISMS
 Spores of clostridia are usually wider than the diameter of the rods in
which they are formed.
 In the various species, the spore is placed centrally, subterminally, or
terminally.
 Most species of clostridia are motile and possess peritrichous flagella.
 A gram stain of a Clostridium species with terminal spores is shown in
Figure 11–2.
CULTURE
 Clostridia are anaerobes and grow under anaerobic conditions.
 a few species are aerotolerant and will also grow in ambient air.
 In general, the clostridia grow well on the blood-enriched media used to grow anaerobes
and on other media used to culture anaerobes as well.

COLONY FORMS
 Some clostridia produce large raised colonies (eg, C perfringens); others produce smaller
colonies (eg, C tetani).
 Some clostridia form colonies that spread on the agar surface. Many clostridia produce a
zone of hemolysis on blood agar.
 C perfringens characteristically produces a double zone of hemolysis around colonies.

GROWTH CHARACTERISTICS

 Clostridia can ferment a variety of sugars.

 many can digest proteins. Milk is turned acid by some and digested by others and
undergoes "stormy fermentation" (ie, clot torn by gas) with a third group (eg, C
perfringens).
 Various enzymes are produced by different species.
ANTIGENIC CHARACTERISTICS
Clostridia share some antigens but also possess specific soluble antigens that permit
grouping by precipitin tests.
 Toxin
 During the growth of C botulinum and during autolysis of
the bacteria, toxin is liberated into the environment.
 Seven antigenic varieties of toxin (A–G) are known.
Types A, B, and E (and occasionally F) are the principal
causes of human illness.
 Types A and B have been associated with a variety of
foods and type E predominantly with fish products.
 The toxin is a 150,000-MW protein that is cleaved into
100,000-MW and 50,000-MW proteins linked by a
disulfide bond.
 Botulinum toxin is absorbed from the gut and binds to
receptors of presynaptic membranes of motor neurons of
the peripheral nervous system and cranial nerves.
 Proteolysis—by the light chain of botulinum toxin—of the target
SNARE proteins in the neurons inhibits the release of
acetylcholine at the synapse, resulting in lack of muscle
contraction and paralysis.
 The SNARE proteins are synaptobrevin, SNAP 25, and syntaxin.
 The toxins of C botulinum types A and E cleave the 25,000-MW
SNAP 25.
 Type B toxin cleaves synaptobrevin.
 C botulinum toxins are among the most toxic substances known.
 The lethal dose for a human is probably about 1–2 g/kg.
 The toxins are destroyed by heating for 20 minutes at 100°C.
 Rare strains of C butyricum and C baratii have also been shown to
produce botulinum neurotoxin and cause botulism in humans.
Those strains that produce toxins E and F are associated with
infant botulism.
Pathogenesis
 Although C botulinum types A and B have been
implicated in cases of wound infection and botulism,
most often the illness is not an infection. Rather, it is
an intoxication resulting from the ingestion of food in
which C botulinum has grown and produced toxin.
 The most common offenders are spiced, smoked,
vacuum-packed, or canned alkaline foods that are
eaten without cooking.
 In such foods, spores of C botulinum germinate;
under anaerobic conditions, vegetative forms grow
and produce toxin.
 In infant botulism, honey is the most frequent vehicle
of infection.
 The pathogenesis differs from the way that adults
acquire infection.
 The infant ingests the spores of C botulinum (or C
butyricum or C baratii), and the spores germinate
within the intestinal tract.
 The vegetative cells produce toxin as they multiply;
the neurotoxin then gets absorbed into the
bloodstream.
 The toxin acts by blocking release of acetylcholine at
synapses and neuromuscular junctions .
 Flaccid paralysis results.
Clinical Findings
 Symptoms begin 18–24 hours after ingestion of the toxic food, with
visual disturbances (incoordination of eye muscles, double vision),
inability to swallow, and speech difficulty; signs of bulbar paralysis are
progressive, and death occurs from respiratory paralysis or cardiac
arrest.
 Gastrointestinal symptoms are not regularly prominent. There is no
fever. The patient remains fully conscious until shortly before death.
 The mortality rate is high. Patients who recover do not develop
antitoxin in the blood.
 In the United States, infant botulism is as common as or more common
than the classic form of paralytic botulism associated with the ingestion
of toxin-contaminated food.
 The infants in the first months of life develop poor feeding, weakness,
and signs of paralysis (floppy baby).
 Infant botulism may be one of the causes of sudden infant death
syndrome.
 C botulinum and botulinum toxin are found in feces but not in serum.
Treatment
 Potent antitoxins to three types of botulinum toxins
have been prepared in horses. Since the type
responsible for an individual case is usually not known,
trivalent (A, B, E) antitoxin must be promptly
administered intravenously with customary
precautions.
 Adequate ventilation must be maintained by
mechanical respirator, if necessary. These measures
have reduced the mortality rate from 65% to below
25%.
 Although most infants with botulism recover with
supportive care alone, antitoxin therapy is
recommended.
Clostridium tetani
CLOSTRIDIUM TETANI
 C tetani, which causes tetanus, is worldwide in distribution in the soil and in the
feces of horses and other animals.
 Several types of C tetani can be distinguished by specific flagellar antigens.
 All share a common O (somatic) antigen, which may be masked, and all produce
the same antigenic type of neurotoxin, tetanospasmin.

 C. tetani is part of a genus of obligate anaerobic, saprophytic, gram-positive

organisms.
 . C. tetani is well known for its toxin-producing ability making it one of the most
dangerous of its genus.
 C. tetani is a spore-forming organism that cannot be eliminated from the
environment and can withstand extreme temperature conditions in both indoor
and outdoor environments.It is well known that tetanus spores can survive in the
environment for many years and are often resistant to heat and disinfectants.
 The source of infection in most people is a wound or a recent injury; the injury is
often mild. In others, tetanus may develop from a burn injury abscess, following a
surgical procedure, IV drug abuse or gangrene. In many cases, the patients are
either under-immunized or have not been vaccinated.
Toxin
 The vegetative cells of C tetani produce the toxin tetanospasmin
(MW 150,000) that is cleaved by a bacterial protease into two
peptides (MW 50,000 and 100,000) linked by a disulfide bond.
 The toxin initially binds to receptors on the presynaptic membranes
of motor neurons.
 It then migrates by the retrograde axonal transport system to the
cell bodies of these neurons to the spinal cord and brain stem.
 The toxin diffuses to terminals of inhibitory cells, including both
glycinergic interneurons and aminobutyric acid-secreting neurons
from the brain stem.
 The toxin degrades synaptobrevin, a protein required for docking of
neurotransmitter vesicles on the presynaptic membrane.
 Release of the inhibitory glycine and -aminobutyric acid is
blocked, and the motor neurons are not inhibited. Hyperreflexia,
muscle spasms, and spastic paralysis result. Extremely small
amounts of toxin can be lethal for humans.
Pathogenesis
 C tetani is not an invasive organism.
 The infection remains strictly localized in the area of
devitalized tissue (wound, burn, injury, umbilical stump,
surgical suture) into which the spores have been introduced.
 The volume of infected tissue is small, and the disease is
almost entirely a toxemia.
 Germination of the spore and development of vegetative
organisms that produce toxin are aided by (1) necrotic tissue,
(2) calcium salts, and (3) associated pyogenic infections, all of
which aid establishment of low oxidation-reduction potential.
 The toxin released from vegetative cells reaches the central
nervous system and rapidly becomes fixed to receptors in the
spinal cord and brain stem and exerts the actions described
above.
Clinical Findings
 The incubation period may range from 4–5 days to as many
weeks.
 The disease is characterized by tonic contraction of voluntary
muscles.
 Muscular spasms often involve first the area of injury and
infection and then the muscles of the jaw (trismus, lockjaw),
which contract so that the mouth cannot be opened.
 Gradually, other voluntary muscles become involved, resulting
in tonic spasms.
 Any external stimulus may precipitate a tetanic generalized
muscle spasm.
 The patient is fully conscious, and pain may be intense.
 Death usually results from interference with the mechanics of
respiration. The mortality rate in generalized tetanus is very
high.
 Prevention & Treatment

• The results of treatment of tetanus are not satisfactory.

Therefore, prevention is all-important.
• Prevention of tetanus depends upon
• (1) active immunization with toxoids; (2) proper care of wounds contaminated with
soil, etc;
• (3) prophylactic use of antitoxin; and
• (4) administration of penicillin.
• The intramuscular administration of 250–500 units of human antitoxin (tetanus
immune globulin) gives adequate systemic protection (0.01 unit or more per
milliliter of serum) for 2–4 weeks.
• It neutralizes the toxin that has not been fixed to nervous tissue. Active
immunization with tetanus toxoid should accompany antitoxin prophylaxis
 Patients who develop symptoms of tetanus should receive muscle
relaxants, sedation, and assisted ventilation.
 Sometimes they are given very large doses of antitoxin (3000–10,000
units of tetanus immune globulin) intravenously in an effort to
neutralize toxin that has not yet been bound to nervous tissue. However,
the efficacy of antitoxin for treatment is doubtful except in neonatal
tetanus, where it may be lifesaving.
 Surgical debridement is vitally important because it removes the
necrotic tissue that is essential for proliferation of the organisms.
Penicillin strongly inhibits the growth of C tetani and stops further
toxin production. Antibiotics may also control associated pyogenic
infection.
 When a previously immunized individual sustains a potentially
dangerous wound, an additional dose of toxoid should be injected to
restimulate antitoxin production. This "recall" injection of toxoid may
be accompanied by a dose of antitoxin if the patient has not had current
immunization or boosters or if the history of immunization is unknown
Control
 Tetanus is a totally preventable disease.
 Universal active immunization with tetanus toxoid should be mandatory.
 Tetanus toxoid is produced by detoxifying the toxin with formalin and
then concentrating it.
 Aluminum-salt-adsorbed toxoids are employed.
 Three injections comprise the initial course of immunization, followed by
another dose about 1 year later.
 Initial immunization should be carried out in all children during the first
year of life.
 A "booster" injection of toxoid is given upon entry into school.
Thereafter, "boosters" can be spaced 10 years apart to maintain serum
levels of more than 0.01 unit antitoxin per milliliter.
 In young children, tetanus toxoid is often combined with diphtheria
toxoid and acellular pertussis vaccine.
 Control measures are not possible because of the wide dissemination of
the organism in the soil and the long survival of its spores.
CLOSTRIDIA THAT PRODUCE INVASIVE INFECTIONS
 Many different toxin-producing clostridia (C perfringens and
related clostridia) can produce invasive infection (including
myonecrosis and gas gangrene) .if introduced into damaged tissue.
 About 30 species of clostridia may produce such an effect, but the
most common in invasive disease is C perfringens (90%).
 An enterotoxin of C perfringens is a common cause of food
poisoning.
Toxins
 The invasive clostridia produce a large variety of toxins and
enzymes that result in a spreading infection.
 Many of these toxins have lethal, necrotizing, and hemolytic
properties.
 The toxin of C perfringens type A is a lecithinase, and its lethal
action is proportionate to the rate at which it splits lecithin (an
important constituent of cell membranes) to phosphorylcholine
and diglyceride.
 The theta toxin has similar hemolytic and necrotizing effects but is not
a lecithinase.
 DNase and hyaluronidase, a collagenase that digests collagen of
subcutaneous tissue and muscle, are also produced.
 Some strains of C perfringens produce a powerful enterotoxin,
especially when grown in meat dishes.
 When more than 108 vegetative cells are ingested and sporulate in the
gut, enterotoxin is formed.
 The enterotoxin is a protein (MW 35,000) that may be a nonessential
component of the spore coat; it is distinct from other clostridial toxins.
 It induces intense diarrhea in 6–18 hours.
 The action of C perfringens enterotoxin involves marked
hypersecretion in the jejunum and ileum, with loss of fluids and
electrolytes in diarrhea.
 Much less frequent symptoms include nausea, vomiting, and fever.
This illness is similar to that produced by B cereus and tends to be self-
limited.
Pathogenesis
 In invasive clostridial infections, spores reach tissue
either by contamination of traumatized areas (soil,
feces) or from the intestinal tract.
 The spores germinate at low oxidation-reduction
potential; vegetative cells multiply, ferment
carbohydrates present in tissue, and produce gas.
 The distention of tissue and interference with blood
supply, together with the secretion of necrotizing
toxin and hyaluronidase, favor the spread of infection.
 Tissue necrosis extends, providing an opportunity for
increased bacterial growth, hemolytic anemia, and,
ultimately, severe toxemia and death
 In gas gangrene (clostridial myonecrosis), a mixed
infection is the rule. In addition to the toxigenic
clostridia, proteolytic clostridia and various cocci and
gram-negative organisms are also usually present.
 C perfringens occurs in the genital tract of 5% of
women. Before legalization of abortion in the United
States, clostridial uterine infections followed
instrumental abortions.
 Clostridium sordellii has many of the properties of C
perfringens. C sordellii has been reported to cause a
toxic shock syndrome after medical abortion.
Endometrial infection with C sordellii is implicated.
 Clostridial bacteremia is a frequent occurrence in
patients with neoplasms.
Clinical Findings
 From a contaminated wound (eg, a compound fracture, postpartum
uterus), the infection spreads in 1–3 days to produce crepitation in
the subcutaneous tissue and muscle, foul-smelling discharge, rapidly
progressing necrosis, fever, hemolysis, toxemia, shock, and death.
 Treatment is with early surgery (amputation) and antibiotic
administration.
 Until the advent of specific therapy, early amputation was the only
treatment. At times, the infection results only in anaerobic fasciitis
or cellulitis.
 C perfringens food poisoning usually follows the ingestion of large
numbers of clostridia that have grown in warmed meat dishes.
 The toxin forms when the organisms sporulate in the gut, with the
onset of diarrhea—usually without vomiting or fever—in 6–18
hours. The illness lasts only 1–2 days.
Treatment
 The most important aspect of treatment is prompt and extensive
surgical debridement of the involved area and excision of all
devitalized tissue, in which the organisms are prone to grow.
 Administration of antimicrobial drugs, particularly penicillin, is
begun at the same time.
 Hyperbaric oxygen may be of help in the medical management
of clostridial tissue infections. It is said to "detoxify" patients
rapidly.
 Antitoxins are available against the toxins of C perfringens,
Clostridium novyi, Clostridium histolyticum, and Clostridium
septicum, usually in the form of concentrated immune
globulins.
 Polyvalent antitoxin (containing antibodies to several toxins)
has been used.
 CLOSTRIDIUM DIFFICILE & DIARRHEAL DISEASE
Pseudomembranous Colitis
 Pseudomembranous colitis is diagnosed by detection of one or both C
difficile toxins in stool and by endoscopic observation of
pseudomembranes or microabscesses in patients who have diarrhea
and have been given antibiotics.
 Plaques and microabscesses may be localized to one area of the bowel.
 The diarrhea may be watery or bloody, and the patient frequently has
associated abdominal cramps, leukocytosis, and fever.
 Although many antibiotics have been associated with
pseudomembranous colitis, the most common are ampicillin and
clindamycin and more recently, the fluoroquinolones.
 The disease is treated by discontinuing administration of the offending
antibiotic and orally giving either metronidazole or vancomycin
 Administration of antibiotics results in proliferation
of drug-resistant C difficile that produces two toxins.
 Toxin A, a potent enterotoxin that also has some
cytotoxic activity, binds to the brush border
membranes of the gut at receptor sites.
 Toxin B is a potent cytotoxin.
 Both toxins are found in the stools of patients with
pseudomembranous colitis.
 Not all strains of C difficile produce the toxins, and
the tox genes apparently are not carried on plasmids
or phage.
Antibiotic-Associated Diarrhea
 The administration of antibiotics frequently leads to
a mild to moderate form of diarrhea, termed
antibiotic-associated diarrhea.
 This disease is generally less severe than the classic
form of pseudomembranous colitis.
 As many as 25% of cases of antibiotic-associated
diarrhea may be associated with C difficile.