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Obligately anaerobic Gram positive endosporeforming rods

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... food poisoning usually derived from meats, and causes S. aureus-like food ... C. botulinum causes botulism, a rare but serious type of food poisoning. ... – PowerPoint PPT presentation

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Title: Obligately anaerobic Gram positive endosporeforming rods


1
Obligately anaerobic Gram positive
endospore-forming rods
  • Clostridium
  • Refer to Introduction to anaerobes

2
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.

3
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

4
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

5
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.

6
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.

7
(No Transcript)
8
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

9
Stormy fermentation in litmus milk media
Double zone hemolysis
10
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.
11
Reverse CAMP Test
Clostridium perfringens
Group B Strep (Test Organism)
Augmented hemolysis
12
Nagler test bottom half contains no anti-toxin
therefore the opaque zone forms. Top half does
contain the anti-toxin
13
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
14
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.

15
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.

16
Flaccid paralysis from botulism
17
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

18
C. botulinum cells note the evident terminal
swelling from sub-terminal endospores
19
The BoTox alternative
20
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?

21
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.

22
tetanus
23
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.

24
C. Tetani note the extreme terminal position of
endospores and exagerated swelling
25
C. tetani colony on SBA
26
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

27
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.

28
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
29
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
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