Corynebacterium diphtheriae Case File

Corynebacterium diphtheriae Case File

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

CASE 7

A 6-year-old girl is brought into the office for evaluation of a sore throat and fever, which she has had for approximately 4 days. Her parents have immigrated to the United States from Russia about 6 months ago. She has not had much medical care in her life, and her immunization status is unknown. On examination the child is anxious, tachypneic, and ill appearing. Her temperature is 38.6°C (101.5°F), and her voice is hoarse. Examination of her pharynx reveals tonsillar and pharyngeal edema with the presence of a gray membrane coating of the tonsil, which extends over the uvula and soft palate. She has prominent cervical adenopathy. Her lungs are clear. You immediately transfer her to the local children’s hospital with the presumptive diagnosis of pharyngeal diphtheria and order confirmatory tests.

◆ What Gram stain characteristics does Corynebacterium diphtheriae have on microscopy?

◆ What factor is required for the expression of diphtheria toxin?

ANSWERS TO CASE 7: Corynebacterium diphtheriae

Summary: A 6-year-old girl who recently arrived from Russia is diagnosed with pharyngeal diphtheria.

◆ Characteristics of C. diphtheriae on Gram stain: The club-shaped appearance of the gram-positive bacillus, often characterized as “Chinese letters” because of adherence of cells following division.

◆ Factor required for the expression of diphtheria toxin: Lysogenic bacteriophage.

CLINICAL CORRELATION

Corynebacteria are ubiquitous in nature and are part of the normal flora of the human respiratory tract and skin. Although most species of Corynebacterium can be opportunistic pathogens, only a few species are commonly associated with human disease. One of those species is Corynebacterium jeikeium, which is most commonly associated with bacteremia and line-related infection in immunocompromised patients. This organism is one of the few species of Corynebacterium that tends to be multidrug resistant. Corynebacterium diphtheriae, the cause of diphtheria, is one of the most pathogenic of the species. Humans are the only known reservoir and transmission is thought to occur by contact with aerosolized droplets, respiratory secretions, or infected skin lesions. Respiratory diphtheria occurs 2–6 days after inhalation of infected droplets. Patients develop nonspecific signs and symptoms of an upper respiratory infection as the organisms multiply locally with in epithelial cells in the respiratory tract. Toxin is then produced eliciting systemic symptoms including fever. An exudate, containing organisms, fibrin, and white and red blood cells, is formed, which is called a pseudomembrane. This grayish membrane covers the tonsils, uvula, and palate and can extend as far as the nasopharynx or larynx. Complications of membrane formation can be respiratory compromise by aspiration of the pseudomembrane, which is a common cause of death in this disease. Symptoms include fever and cervical lymphadenopathy (bull neck).

Cutaneous diphtheria, although rare in the United States, occurs from invasion of the organism from the patient’s skin into the subcutaneous tissue. A papule develops at the site of contact that later becomes covered by a grayish membrane. As in respiratory diphtheria, toxin production by the organism elicits a systemic response with fever. Diphtheria toxin can also have effects on the heart (myocarditis) and nervous system (dysphagia, paralysis).

APPROACH TO SUSPECTED DIPHTHERIA

INFECTION

Objectives

1. Know the characteristics and virulence factors of C. diphtheriae.
2. Know the factors involved with the expression of and the mechanism of action of the C. diphtheriae exotoxin.

Definitions

Lysogenic bacteriophage: Virus that infects bacteria.

Elek Test: An immunodiffusion test to detect the production of diphtheria toxin in a strain of C. diphtheria.

Pseudomembrane: Membrane formed in diphtheria, which consists of dead cells, leukocytes, and fibrin.

DISCUSSION

Characteristics of Corynebacterium diphtheriae

Corynebacterium diphtheriae is a nonencapsulated gram-positive bacillus. It is nonmotile, non-spore-forming, and club shaped. The cells often remain attached after division and form sharp angles, giving a characteristic “Chinese letter” appearance on microscopy.

Corynebacterium diphtheriae is divided into three subtypes—gravis, intermedius, and mitis—based on colony morphology and biochemical testing.

In the presence of a lysogenic β-phage, C. diphtheriae can produce a highly potent exotoxin. The toxin, which is the major virulence factor of this organism, consists of two components. The B segment binds to specific receptors on susceptible cells. Following proteolytic cleavage, the A segment is released into the host cell, where it can inhibit protein synthesis. The exotoxin targets a factor present in mammalian cells but not in bacterial cells, thus causing host tissue damage without affecting bacterial replication. Toxin-related tissue necrosis causes the characteristic pseudomembrane seen in clinical diphtheria.

Diagnosis

The differential diagnosis in the presence of sore throat, fever, and cervical lymphadenopathy would include streptococcal pharyngitis and infectious mononucleosis.

Clinical diagnosis of diphtheria can be made by visualization of the characteristic pseudomembrane formation. The membrane should not be removed because of the tight adherence to the epithelial surface and the chance for subsequent bleeding. Cultures should be collected from the throat or nasopharynx. A Gram stain would reveal the characteristic gram-positive club-shaped bacilli.

Corynebacterium with the exception of a few lipophilic species will grow well on most nonselective media within 24 hours. Colonies are usually nonpigmented and small, without hemolysis on blood agar. However, C. diphtheriae is more fastidious and specimens should be plated on a selective medium such as Tellurite in addition to the nonselective media. Colonies of C. diphtheriae will appear black on Tellurite media. Colonies growing on Loeffler media can be stained with methylene blue to observe the characteristic metachromatic granules. Definitive identification is made by biochemical tests usually performed at a reference or state public health laboratory, where the isolate will be further tested for toxin production. The Elek test is an immunodiffusion assay for detection of production of C. diphtheria toxin by the isolate.

Treatment and Prevention

Therapy for diphtheria is a combination of antimicrobial therapy (erythromycin) and antitoxin. The antitoxin must be administered rapidly, before the toxin binds to epithelial cells. Diphtheria can be prevented by vaccination with diphtheria toxoid (DPT). Infected patients should be isolated from other susceptible persons to prevent secondary spread of the disease. Prophylaxis with erythromycin can also be given to close contacts who are at risk.

COMPREHENSION QUESTIONS

[7.1] The mechanism of action of the exotoxin produced by C. diphtheriae can be characterized by which of the following?

A. Acting as a superantigen that binds to MHC class II protein and the T-cell receptor.

B. Blocking the release of acetylcholine causing anticholinergic symptoms.

C. Blocking the release of glycine (inhibitory neurotransmitter).

D. Inhibits protein synthesis via EF-2 adenosine diphosphate (ADP) ribosylation.

E. Stimulation of adenylate cyclase by ADP ribosylation of G-protein.

[7.2] Which of the following most accurately describes the therapy available for the prevention and treatment of C. diphtheriae?

A. Antimicrobial therapy for prophylaxis only

B. Antimicrobial therapy and prophylaxis, antitoxin, and toxoid (DPT)

C. Antitoxin only

D. Diphtheria toxoid (DPT) booster vaccination only

Answers

[7.1] D. Corynebacterium diphtheriae produces a potent exotoxin encoded by a lysogenic β-prophage. Following proteolytic cleavage, the A segment is released into the host cell where it inhibits only mammalian protein synthesis (ribosomal function) via ADP ribosylation of EF-2. Inhibition of protein synthesis disrupts normal cellular physiologic functions that are believed to be responsible for the necrotizing and neurotoxic effects of diphtheria toxin. An exotoxin with a similar mode of action is produced by some Pseudomonas aeruginosa strains. Staphylococcus aureus is responsible for producing the toxic shock syndrome toxin that acts as a superantigen leading to T-cell activation. Clostridium tetani blocks the release of glycine, leading to “lock-jaw.” Clostridium botulinum blocks the release of acetylcholine, causing central nervous system (CNS) paralysis and anticholinergic symptoms. Finally, the heat-labile toxin produced by Escherichia coli causes watery diarrhea by stimulating adenylate cyclase.

[7.2] B. Protection against C. diphtheriae can be established through both active and passive immunity. Active immunity consists of a toxoid administered in the form of the DPT vaccine. Passive immunity is established by administering diphtheria antitoxin (immunoglobulins). Antimicrobial therapy (erythromycin) can be used to effectively treat patients with clinical diphtheria.

MICROBIOLOGY PEARLS ❖ The club-shaped appearance of the gram-positive bacillus C. diphtheriae is often characterized as “Chinese letters” as a result of adherence of cells following division. ❖ The typical clinical feature of diphtheria is pseudomembrane formation. ❖ Diphtheria is preventable by administration of DPT vaccine, which provides immunity for diphtheria, pertussis, and tetanus.

REFERENCES

Holmes, RK. Biology and molecular epidemiology of diphtheria toxin and the tox gene. J Infect Dis 2000;181(suppl 1):S156–67. 

Murray PR, Rosenthal KS, Pfaller MA. Corynebacterium and other gram-positive bacilli. In: Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology, 5th ed. St. Louis, MO: Mosby, 2005:279–85.

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