CASE 6

A 52-year-old man presents for the evaluation of diarrhea and abdominal pain, which have
been worsening over the past week. He is now having 8 to 10 watery stools a day and mild
cramping pain. He denies vomiting, fever, ill contacts, or having had blood in his stool. He
has no history of gastrointestinal diseases. He states that approximately 10 days ago he
completed a course of amoxicillin/clavulanate for pneumonia. On examination he is mildly ill
appearing, but his vital signs are normal. His abdomen is soft, has hyperactive bowel sounds,
and is diffusely, mildly tender. A stool sample is negative for blood but positive for
leukocytes. A stool culture is negative, but a specific toxin assay is positive.

What is the most likely etiologic agent of this disease?

Which condition predisposes this organism to cause disease in humans?

ANSWERS TO CASE 6:
Clostridia
Summary: A 52-year-old man who recently took oral antibiotics now has diarrhea. Fecal
leukocytes are present in the stool, and a toxin test is positive.

• Most likely etiologic agent: Clostridium difficile

• Condition predisposing disease in humans: Recent antibiotic exposure

CLINICAL CORRELATION
There are approximately 90 bacterial species of Clostridium, about 20 of which are known to
cause disease in humans. They are found widely in soil, decaying vegetation, and the intestinal
tracts of humans and other vertebrates. Infection occurs in patients with predisposing factors
including trauma, surgery, immunosuppression, and prior treatment with antibiotics. C.
perfringens is the most common Clostridium species isolated from human infections and is a
cause of wound infections including gas gangrene. C. tetani is associated with the toxin-mediated
disease, tetanus, which occurs in unvaccinated persons who come in contact with the organism.
The spores of the organism survive for long periods of time in the soil and are introduced into the
person following deep puncture wounds. Tetanus is characterized by tonic spasms usually
involving the muscles of the neck, jaw (lockjaw), and trunk. C. botulinum is the causative agent of
botulism. Botulism occurs when spores are consumed, usually from improperly canned
vegetables. Symptoms of nausea, blurred vision, and weakness of the upper extremities spreading
downward occur within 12 to 36 hours after ingestion of the toxin. Infant botulism is associated
with consumption of honey.
C. difficile can be isolated in the stool of fewer than 5% of healthy adults; however, the stools
of up to 70% of healthy infants are infected with the organism. Most cases of C. difficile colitis
occur during or following a course of antibiotics. Antibiotics alter the intestinal flora allowing for
an overgrowth of C. difficile, which either already exists in the intestinal tract or is introduced
from an environmental source. Disease can range from asymptomatic carriage of the organism to
mild diarrhea to pseudomembranous colitis, which can be further complicated by toxic megacolon
and bowel perforation.

APPROACH TO:
Suspected Clostridium Difficile Infection
OBJECTIVES
1. Know the characteristics of the Clostridium species.
2. Know the virulence factors and diseases associated with Clostridium bacteria.

DEFINITIONS
Antibiotic-associated diarrhea: Gastroenteritis caused by C. difficile

Pseudomembranous colitis: Presence of nodules or plaques on erythematous (red) colonic

mucosa seen by sigmoidoscopy, associated with C. difficile colitis

DISCUSSION
Characteristics of Clostridium difficile
C. difficile is an anaerobic, spore-forming, toxigenic gram-positive rod. Some strains have a
thin capsule and some have fimbriae, although the significance of these is uncertain. C. difficile,
so named because of the initial difficulty in isolating and culturing the organism, requires a
selective medium for growth which also inhibits normal stool flora.
The virulence factors of C. difficile include toxin production as well as production of other
enzymes, such as hyaluronidase. Toxin A is an enterotoxin, and toxin B, the more biologically
active toxin in humans, is a cytotoxin. The specific role each component plays in disease in
humans is unknown. The enterotoxin is chemotactic and initiates the release of cytokines,
hypersecretion of fluids in the intestinal tract, and hemorrhagic necrosis. Depolarization of actin
microfilaments occurs, which leads to destruction of the cellular cytoskeleton disruption of tight
junctions between epithelial cells. A new strain of C. difficile has been recently identified, which
is more virulent and more likely to cause megacolon. This strain has been found to produce larger
quantities of toxins A and B in addition to a new toxin, binary toxin. Formation of spores allows
the organism to survive under stressful situations in the environment for extended periods of time.
Spore formation also allows the organisms to survive in the hospital environment and to be
transferred from patient to patient on fomites.

DIAGNOSIS
Antibiotic-associated diarrhea is the most common cause of diarrhea that develops in patients
who have been hospitalized for 3 or more days. Clinical diagnosis can be made by visualization of
the pseudomembrane (fibrin, bacteria, cell debris, white blood cells).
The gold standard for laboratory diagnosis of antibiotic-associated diarrhea caused by C.
difficile is detection of toxin production in the stool using a tissue culture assay, where a
specific antibody neutralizes the toxin and, therefore, the production of cytopathic effect.
However, this assay requires tissue culture facilities as well as approximately 3 days for
completion. Culture of C. difficile can be performed on selective media, Cycloserine, Cefoxitin,
and Fructose Agar in an egg yolk agar base (CCFA medium), in an anaerobic environment. After
24–48 hours, incubation colonies will fluoresce chartreuse on CCFA and have a barnyard odor.
Specific identification can be made using commercially available rapid methods that detect fatty
acids produced by the organism or by gas/liquid chromatography. Growth of the organism would
have to be followed up by detection of toxin for a specific diagnosis of disease.
Commercially available membrane or microwell-based enzyme immunoassays are available
for rapid detection of toxin A or toxin A and B in a stool specimen. For optimal recovery testing
of three stools on 3 days is recommended.

TREATMENT AND PREVENTION

The first-line treatment for C. difficile disease is oral metronidazole, with oral vancomycin
reserved for those who fail first-line treatment. For severe disease oral vancomycin is usually
superior in terms of better cure rates, less treatment failures, and lower relapse rates.
Unfortunately, relapse can occur in 20% to 30% of adequately treated patients because of the
resistance of the spores to treatment. A second round of treatment is usually successful. Failure is
not usually attributed to resistance of the organism to vancomycin or metronidazole. Prevention of
C. difficile in hospitalized patients involves good infection control procedures that include
isolation of the infected patient. No treatment is required in asymptomatic carriers. Patients with
recalcitrant infections may benefit from stool transplantation from family members who are
asymptomatic and not infected.

COMPREHENSION QUESTIONS
6.1 A 24-year-old woman was hospitalized for acute pelvic inflammatory disease, and was treated
with intravenous cefoxitin and clindamycin. Three days after discharge from the hospital, she
developed severe abdominal pain and diarrhea. Which of the following organisms is the most
likely causative agent?
A. Bacillus anthracis
B. Bacillus cereus
C. Clostridium botulinum
D. Clostridium difficile
E. Clostridium tetani
6.2 Which laboratory test is the best method to confirm the diagnosis of the patient in Question
6.1?
A. Gas/liquid chromatography
B. Pseudomembranous visualization
C. Rapid fatty acid detection assays
D. Tissue culture toxin detection assay
6.3 A hospitalized patient developed severe diarrhea and pseudomembranous colitis within 5 days
after antibiotic therapy was initiated. The severe diarrhea and pseudomembranous colitis
occurred as a result of which of the following?
A. Collagenase
B. Fibrinolysin
C. Hyaluronidase
 D. Lecithinase
E. Mucinase
F. Toxins A and B

ANSWERS
6.1 D. The use of broad-spectrum antibiotics such as ampicillin and clindamycin has been
associated with pseudomembranous colitis. Antibiotic administration results in the
proliferation of drug-resistant C. difficile that produces toxins A (a potent enterotoxin with
cytotoxic activity) and B (a potent cytotoxin). This disease is most appropriately treated by
discontinuing the offending antibiotic and instead administering oral doses of either
metronidazole or vancomycin. The administration of antibiotics may also lead to a milder form
of diarrhea, called antibiotic-associated diarrhea. This form is associated with C. difficile
approximately 25% of the time.
6.2 D. All of the above tests may be used as detection assays for C. difficile. However, only the
tissue culture toxin detection assay is the gold standard laboratory test. This test involves a
specific toxin-neutralizing antibody that detects toxin (toxins A and B) production in the stool
using a tissue culture detection assay. Not all C. difficile strains produce toxins, and the toxin
genes are not carried on plasmids or phages.
6.3 F. Clostridium difficile produces 2 toxins, toxins A and B. Both toxins are present in stool
samples. Toxin A is enterotoxic causing the severe diarrhea, whereas toxin B is cytotoxic
leading to the destruction of enterocytes, resulting in pseudomembranous colitis. For
additional information please refer to the discussions for questions 6.1 and 6.2.

 MICROBIOLOGY PEARLS

The most common cause of diarrhea in a patient who has been hospitalized for 3 or more
days is C. difficile.
The initial treatment for pseudomembranous colitis is metronidazole. Oral vancomycin is
used for those who fail to respond to metronidazole.
Detection of toxins in the stool is the method of choice for diagnosis of C. difficile colitis.

REFERENCES
Allen SD, Emery CL, Lyerly DM. In: Murray PR, Baron EJ, Jorgensen JH, et al., eds. Manual of
Clinical Microbiology. 8th ed. Washington, DC: ASM Press; 2003:835-856.
Murray PR, Rosenthal KS, Pfaller MA. Clostridium. In: Murray PR, Rosenthal KS, Pfaller MA.
Medical Microbiology. 5th ed. St. Louis, MO: Mosby; 2005:401-420.