Obligately anaerobic Gram positive endosporeforming rods

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Obligately anaerobic Gram positive
endospore-forming rods

• Clostridium
• Refer to Introduction to anaerobes

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Clostridium Introduction

• Clostridia are obligately anaerobic
  endospore-forming Gram positive rods there is
  your presumptive ID. Because of their spores,
  Clostridia cannot be as easily destroyed as most
  other microbes. Only autoclaving, incineration,
  radiation and antibiotics (fortunately fairly
  susceptible) are effective.
• Clostridia are ubiquitous in the soil and water
  worldwide, and therefore we come in contact with
  them daily. Fortunately, as obligate anaerobes,
  the conditions necessary for proliferation to
  disease-causing infection is not commonly present
  in non-sterile tissues such as respiratory or
  digestive tract. C. difficile is unique it is a
  GI tract pathogen, but only flourishes there when
  transmitted nosocomially following extended
  antibiotic therapy which decimates the normal gut
  flora.

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continued

• Given the opportunity, some species can
  proliferate and cause various pathology using a
  battery of exotoxins, some of which are the most
  potent toxins known to science.
• Human pathogenic species include C. perfringens,
  C. tetani, C. botulinum, and C. difficile. As
  said before, C. difficile is unique among these
  species.
• For the most part, penicillin is effective
  against Clostridial infections

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Clostridium Presumptive ID

• Clostridia are obligately anaerobic
  endospore-forming Gram positive rods
• Some have a tendency to easily over-decolorize,
  especially if a very young culture is not used
  for smear preparation.
• Endospores may or may not be apparent on the Gram
  stain. Increasingly aerobic conditions prevent
  endospore formation in the Clostridia, whereas
  decreasing aerobiosis limits endospore production
  in Bacillus species. Confusion between these
  genera are not uncommon.
• If endospores are not apparent the culture should
  be subjected to the heat-shock test or the
  ethanol tolerance test. Only endospore producers
  can withstand these adverse conditions
• Wet-mounts may be superior to Gram stains to see
  endospores

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Clostridium perfringens

• Text C. perfringens is by far the most commonly
  isolated species from human sources. But what
  about C. difficile? C. perfringens causes 2
  primary conditions. One is a relatively mild but
  common food poisoning usually derived from meats,
  and causes S. aureus-like food poisoning
  symptoms.
• The other is gas gangrene (ie. myonecrosis).
  Gangrene means death of tissue. Cells are
  introduced into flesh via traumatic implantation
  (historically a lot from war wounds) or surgical
  incision. Under anaerobic conditions they
  multiply, fermenting body substances producing
  gas causing tissue disruption.
• Mortality (often within 2 days) was high prior to
  better treatment including use of anti-toxin,
  surgical debridement, and widespread use of
  antibiotics (pennicillin). Death occurs via
  septic shock and numerous complications.

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continued

• Lecithinase (alpha toxin) is the primary
  exotoxin disrupts membranes and proteins
  hemolysis and tissue necrosis, especially of
  muscle connective tissue (vessels, etc). RBCs
  are disrupted anemia, jaundice, and
  blood-tinged exudates. Gas pressure and
  disrupted tissues crepitation (popping sounds)
  and compromised barriers. Amputation was common
  in war.

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C. perfringens ID

• C. perfringens is the easiest Clostridium to
  speciate due to a unique combination of
  characteristics
• Anaerobic endospore-forming Gram positive rod (ID
  to Genus level)
• Short fat square-ended rod with no apparent
  endospore in the Gram stain
• Double zone hemolysis on SBA (only one in genus)
• Positive reverse CAMP test with S. agalactiae
  (97 C. perfringens)
• Lecithinase positive - Nagler test conducted
  using egg yolk agar with anti-lecithinase
• Stormy fermentation in litmus milk due to acid
  and gas production

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Stormy fermentation in litmus milk media
Double zone hemolysis
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reverse CAMP result of C. perfringens (a, b c)
Called reverse CAMP because S. agalactiae is
the primary or central streak in this case rather
than S. aureus.
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Reverse CAMP Test
Clostridium perfringens
Group B Strep (Test Organism)
Augmented hemolysis
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Nagler test bottom half contains no anti-toxin
therefore the opaque zone forms. Top half does
contain the anti-toxin
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Nagler Test
C.perfringens band streaked on Egg Yolk Agar
Antitoxin placed on right half of plate
before inoculation and incubation
Cloudy precipitate caused by lecithinase produced
by C. perfringens
Cloudy precipitate does not form
because lecithinase is neutralized by
anti-lecitinase
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C. botulinum

• C. botulinum causes botulism, a rare but serious
  type of food poisoning. It has been historically
  associated with canned foods, either home canned
  or industrially. Other more commonly affected
  foods include home-cured ham, fermented fish,
  canned fruits (cranberries), and honey. A few
  recent cases involved native Alaskans eating
  whale meat.
• The toxin, botulin or botulinum toxin, is the
  most potent toxin known. A mere trace is
  sufficient to cause paralysis and death. Like C.
  diptheriae, only C. botulinum cells lysogenized
  by a bacteriaphage can produce the toxin.
• Botulin attaches to the neuromuscular junction of
  affected nerves preventing the release of
  acetylcholine causing flaccid paralysis. Death
  can occur within 2 hours due to smooth and/or
  cardiac muscle paralysis. Alternatively, symptom
  onset may not occur for a week.

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continued

• Besides weakness and paralysis, double vision,
  impaired speech and difficulty in swallowing
  frequently occur
• Infant botulism, unlike that in adults, follows
  ingestion of C. botulinum endospores, most
  commonly from honey. Lack of an established
  intestinal microbial community allows the
  organism to grow in the infant colon. Although
  rare, infant botulism is now the predominant
  type.
• Fortunately, anti-toxin therapy results in
  complete recovery of all affected patients.

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Flaccid paralysis from botulism
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C. botulinum ID Bailey Scott

• C. botulinum is culturable, usually on AnBAP, but
  is not commonly cultured. Cells are usually seen
  in uneaten food.
• Cells appear club shaped or raquet-shaped due to
  terminal swelling from sub-terminal endospores.
  Compared to C. tetani, diameter of swelling is
  greater in a plane parallel to the cell.
• Spores may be evident in Gram stained smears or
  wet mounts.
• Diagnosis via clinical presentation and
  demonstration of toxin in serum, stool or other
  GI sample, or in uneaten food

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C. botulinum cells note the evident terminal
swelling from sub-terminal endospores
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The BoTox alternative
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C. tetani

• C. tetani causes tetanus, a rare (here now) but
  frequently fatal neurological condition much
  like, but the opposite of botulism. The potent
  neruotoxin, tetanospasmin, inhibits release of
  neurotransmitters from neural synapse resulting
  in muscular rigidity via spastic paralysis.
  How exactly it does this is arguable. The Greek
  tetanos to stretch.
• As in botulism, skeletal muscles are affected
  first (one of the 1st is the maseter trismus or
  lockjaw), but death results from smooth and/or
  cardiac muscle paralysis.
• Infection results from introduction of spores,
  usually from the soil where they are common. One
  means of introduction is traumatic implantation,
  which is often occupationally related, or just
  working (or playing) around the yard. Rust has
  NOTHING to do with it, OK?

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C. tetani

• Most cases worldwide are infants who contract the
  infection through the umbilical stump, either
  accidentally or from dung slapped on the
  umbilicus as part of a ritualistic ceremony
• Most cases in the US (only 5/yr on avg) are in
  folks over 60 yrs age, maybe due to time since
  vaccination? Mortality rates run 60 overall,
  andlt 30 in the US.
• Other symptoms include headache, difficulty
  swallowing, spasms, and sweating. Patients are
  extremely irritable.
• Tetanus is the T in the DPT vaccine. It is a
  conjugated vaccine just like the other 2. A
  booster shot is required every ten years. The
  toxoid is the anti-toxin in this case.

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tetanus
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C. tetani ID

• C. tetani is culturable, usually on AnBAP, but is
  not commonly cultured. Cells are recoverable
  from wounds in 1/3 of cases.
• Cells are similar to those of C. botulinum but
  prevalence of cells with spores in a given smear
  may be more scarce. Spores are extreme terminal
  and appear bulbous with exaggerated swelling.
  Compared to C. botulinum, diameter of swelling is
  greater in a plane perpendicular to the cell.
• Spores may be evident in Gram stained smears or
  wet mounts.
• Diagnosis via clinical presentation and serology.
  Additional clinical evidence is generally absent
  and of little value.

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C. Tetani note the extreme terminal position of
endospores and exagerated swelling
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C. tetani colony on SBA
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Clostridium difficle

• C. difficile is a common cause of diarrhea in
  hospitalized patients (it is nosocomial,
  fecal-oral) undergoing antimicrobial therapy (for
  gt 4 days). Statistically, 30 of hospitalized
  patients become infected and 1/3 of these develop
  diarrhea. It is also thought to be present in
  the colon of gt30 of neonates. So C. perfringens
  is the most common Clostridia?
• A complication of C. difficile diarrhea is a
  serious condition called antibiotic-associated
  pseudomembrane enterocolitis (PMC). Antibiotics
  kill off much of the normal gut flora giving this
  highly resistant organism an opportunity to
  proliferate.
• C. difficile produces two potent toxins an
  enterotoxin causing water loss and diarrhea (like
  cholera) and a cytotoxin causing pseudomembrane
  formation

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Clostridium difficle

• Therapy includes discontinuation of the original
  antibiotic (often ampicillin) allowing the gut
  flora to re-establish and out-compete the
  pathogen. Severe cases require use of
  vancomycin.
• Reported mortality rates (10-30) are exaggerated
  by the compromised status of the host.
• C. difficile can be isolated on a selective agar
  medium (Cycloserine-Cefoxitin-Fructose Agar or
  CCFA), but this is generally not recommended
  because C.difficle can be isolated from healthy
  people and not all strains isolated are toxigenic
• Diagnosis is by clinical presentation and by
  serological confirmation of the toxins. An ELISA
  test is available.

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Identification of Clostridium
Anaerobic, Fat, Square-ended Gram Positive Rods
Double Zone Hemolysis
Yes
No
Heat Resistant or Ethanol Tolerant
Clostridium perfringens
Yes
No
Lactobacillus Eubacterium
Aerotolerant
Yes
C. tertium

Confirm terminal, sub-terminalSpores
No
C. botulinum, C. tetani Check clinical history,
send to reference lab
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Presumptive Identification
Gram Stain
Pos Rod
Pos Cocci
Neg Coccus
Neg Rod
Yes
No
Spore
Peptostrepto- coccus
Bacteroides Fusobacterium Porphyrmonas Prevotella
Clostridium
Veillonela
Actinomyces (Branching) Bifidobacterium
(Bifurcations) Lactobacillus (regular, some
chains) Eubacterium (regular no
chains) Propionibacterum (diphtheroid)
Check Heat Resistance or Ethanol Tolerance