Clostridia Case File

Clostridia Case File

Eugene C.Toy, MD, Cynthia Debord, PHD, Audrey Wanger, PHD, Gilbert Castro, PHD, James D. Kettering, PHD, Donald Briscoe, MD

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 8–10 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. Clostridium perfringens is the most common Clostridium species isolated from human infections and is a cause of wound infections including gas gangrene. Clostridium 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. Clostridium 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 12–36 hours after ingestion of the toxin. Infant botulism is associated with consumption of honey.

Clostridium difficile can be isolated in the stool of fewer than 5 percent of healthy adults; however, up to 70 percent of healthy infants have the organism in their stool. Most cases of C. difficile colitis occur during or after 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

Clostridium 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. Clostridium 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 can 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. Unfortunately, relapse can occur in 20–30 percent 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.

COMPREHENSION QUESTIONS

[6.1] Which organism listed below may cause a life-threatening gastroenteritis as a result of use of a broad spectrum antimicrobial agent?

A. Bacillus anthracis

B. Bacillus cereus

C. Clostridium botulinum

D. Clostridium difficile

E. Clostridium tetani

[6.2] Clostridium difficile, as the causative agent in antibiotic-associated diarrhea, can best be detected using which of the following gold standard laboratory tests?

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. Toxin 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 Toxin A (a potent enterotoxin with cytotoxic activity) and Toxin B (a potent cytotoxin). This disease is best treated by discontinuing the use of the offending antibiotic and administering oral doses of metronidazole or vancomycin. Administration of antibiotics may also lead to a milder form of diarrhea, called antibioticassociated diarrhea. This form is associated with C. difficile about 25 percent 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 (Toxin A and B) production in the stool using a tissue culture detection assay. Not all C. difficile strains produce toxins, and the tox genes are not carried on either plasmids or phages.

[6.3] F. Clostridium difficile produces two 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, 2003:835–56. Murray PR, Rosenthal KS, Pfaller MA. Clostridium. In: 

Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology, 5th ed. St. Louis, MO: Mosby, 2005:401–20.

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