Case 1 introduction

Infection with C. trachomatis can be rapidly diagnosed by detection of the bacterial nucleic acid in patient samples, collected using a cotton swab, from the vagina, urethra, or conjunctiva. Diagnostic tests for nucleic acid detection include PCR amplification or direct DNA hybridization assays.

Currently, the best method of preventing chlamydial infection is education and proper sanitation. Ocular infection of Chlamydia trachomatis can sometimes but not always be prevented by administration of topical tetracycline drops. It is the lack of this antibiotic in underdeveloped countries that makes Chlamydia trachomatis prevalent in these areas. C. trachomatis, C. psittaci, and C. pneumonia are all treated with tetracycline or erythromycin. Azithromycin is effective for cervicitis and urethritis. Pelvic inflammatory disease is treated with ceftriaxone and 2 weeks of doxycycline.

[5.1] A 32-year-old immigrant woman from Tanzania delivers a healthy baby boy. Because this woman had no regular doctor, no preliminary tests were performed prior to delivery. Thirteen days after delivery, the child develops swelling of both eyes with the presence of a yellow discharge. The presence of intracytoplasmic inclusion bodies is detected. Which antibiotic would be most appropriate in this situation?

[5.2] What diagnostic test is best to identify an infection with Chlamydia trachomatis?

[5.3] A 29-year-old bird collector presents to the local clinic with what he describes as flu-like symptoms. He doesn't look ill, and has a slight fever, headache, and a dry hacking cough. He denies the production of sputum or hemoptysis. There are no crackles heard on auscultation, and a radiograph shows small streaks of infiltrate. It is determined that he has pneumonia. What is the most likely organism causing his disease?

[5.1] E. The symptoms described are classic for inclusion conjunctivitis caused by Chlamydia trachomatis. The infection was most likely passed from the mother to child during vaginal delivery. The infection usually presents 2 weeks after delivery and is characterized by swollen eyes and a yellow discharge. The drug of choice for this infection is erythromycin eyedrops. Most children are given erythromycin eye drops prophylactically postbirth. Tetracyclines are not given to young children due to staining of teeth.

[5.2] C. The most specific test used to detect a chlamydial infection is a DNA probe. Chlamydia trachomatis is a gram-negative obligate intracellular parasite, and any blood or urine culture would not be helpful for diagnosis. C. trachomatis is not present in stool. Darkfield microscopy is used to view spirochetes, which C. trachomatis is not.

[5.3] B. Although all of the organisms listed above cause pneumonia, only two of them are atypical. Atypical pneumonia is characterized by a dry hacking cough, fever, and headache. These include Chlamydia psittaci and Chlamydia pneumoniae. The mention of birds should point you in the direction of C. psittaci, because they are the reservoir for the organism that is inhaled through dry feces. Typical pneumonias are characterized by hemoptysis of pus-laden sputum, and patients appear very sick. The typical pneumonias include streptococcal infections.

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 about 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?

 Most likely etiologic agent: Clostridium difficile.

 Condition predisposing disease in humans: Recent antibiotic exposure.

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

CLINICAL CORRELATION

Introduction

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 12-36 hours after ingestion of the toxin.

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. 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.

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. However, this assay requires tissue culture facilities as well as approximately three 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. Colonies in 24-48 hours 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 three days is recommended.

Treatment and Prevention

Treatment is with oral vancomycin or metronidazole. 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 patient.

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

[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?

[6.3] A hospitalized patient developed severe diarrhea and pseudomembranous colitis within five days after antibiotic therapy was initiated. The severe diarrhea and pseudomembranous colitis occurred as a result of which of the following?

[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 Clostridium 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 antibiotic-associated 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, toxin 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.

A 6-year-old girl is brought into the office for evaluation of a sore throat and fever, which she has had for about 4 days. She is the daughter of parents who 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 C. diphtheriae have on microscopy?

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

 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.

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

CLINICAL CORRELATION

Introduction

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 C. 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. C. 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 to 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

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.

Objectives

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

Discussion

Characteristics of C. diphtheriae

C. diphtheriae is a non-encapsulated 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.

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

In the presence of a lysogenic beta-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.

The differential diagnosis with 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 with in 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's 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.

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

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

[7.1] D. C. diphtheriae produces a potent exotoxin encoded by a lysogenic -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 aeruginos a strains. S. 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 E. 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.
CASE 8

INTRODUCTION

A 72-year-old female nursing home resident is transferred to the hospital because of fever and altered mental status. She has advanced Alzheimer disease, is bed bound, and has an indwelling Foley catheter as a consequence of urinary incontinence. Her baseline mental status is awake and talkative, but oriented only to person. In the hospital now, she has a temperature of 38.3C (101F) and tachycardia (a rapid heart rate). She mumbles incoherently and is otherwise nonverbal. Her skin is cool, dry, and without ulceration. Her mucous membranes are dry. Her abdomen is soft, has normoactive bowel sounds, and is apparently tender in the suprapubic region. Her urinary catheter is draining cloudy urine. A urinalysis reveals too numerous to count white blood cells and bacteria. Gram stain of the urinary sediment reveals gram-positive cocci. Blood and urine cultures also grow gram-positive cocci.

 What is the most likely cause of this infection?

 How does this organism acquire antibiotic resistance?