Bacillus anthracis Case File

Bacillus anthracis Case File

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

CASE 1

A 53-year-old male farmer presents for evaluation of a growth on his arm. About a week ago, he noticed some mildly itchy red bumps on his arm. They started to blister a day or two later and then ruptured. During this time he had a low-grade fever, but otherwise felt well. Further questioning reveals that he has had no ill contacts and never had anything like this before. He has cows, horses, goats, sheep, and chickens on his farm. On examination of his right upper arm, you find a 4.5-cm circular black eschar surrounded by several vesicles (blisters) and edema. He has tender axillary lymph node enlargement (adenopathy). A Gram stain of fluid drained from a vesicle and a biopsy from the eschar both show chains of gram-positive bacilli on microscopy.

◆ What organism is the likely cause of this disease?

◆ What are the primary virulence factors of this organism?

ANSWERS TO CASE 1: Bacillus anthracis

Summary: A 53-year-old male farmer has a 4.5 cm circular skin lesion of black eschar surrounded by vesicles and edema. The Gram stain on microscopy shows gram-positive bacilli in chains.

◆ Organism most likely causing disease: Bacillus anthracis

◆ Primarily virulence factors: Capsular polypeptide and anthrax toxin

CLINICAL CORRELATION

Bacillus anthracis is the etiologic agent of cutaneous, gastrointestinal, and inhalational anthrax. Approximately 95 percent of anthrax disease is cutaneous. Bacillus anthracis is distributed worldwide, and all animals are susceptible, but it is more prevalent in herbivores. Infected animals often develop a fatal infection and contaminate the soil and water with B. anthracis that can sporulate and continue to survive in the environment for many years. Oxygen is required for sporulation, and the spores will grow on culture plates, in soil, or in the tissue of dead animals. Human infections are caused by either penetration of these spores through the skin barrier (cutaneous), ingestion of the spores (rare), or inhalation of the spores (so-called wool-sorters’ disease), which usually occurs while processing animal products. Person-to-person transmission of anthrax has not been described. Cutaneous anthrax, the most common clinical manifestation, occurs within 2–3 days of exposure to an infected animal or animal product. A papule develops at the site of inoculation, which progresses to form a vesicle. A characteristic black eschar is formed after rupture of the vesicle and development of necrosis in the area. In rare cases the disease progresses and becomes systemic with local edema and bacteremia, which can be fatal if untreated.

The only other Bacillus species frequently associated with human disease is B. cereus, which is a cause of gastroenteritis following ingestion of a contaminated food product, most commonly fried rice. The spores of B. cereus can also survive in the soil and be responsible for traumatic wound infections, particularly to the eye, when soil contamination is involved.

APPROACH TO SUSPECTED ANTHRAX INFECTION

Objectives

1. Know the structure and characteristics of B. anthracis.
2. Know the clinical diseases caused by and virulence of B. anthracis.
3. Know the structure and characteristics of B. cereus.
4. Know the clinical diseases caused by and virulence of B. cereus.

Definitions

Eschar: Skin lesion associated with cutaneous anthrax and resembling a black, necrotic sore.

Wool-sorters’ disease: Disease associated with inhalation of anthrax spores from infected animal products, most often associated with sheep wool.

Differential diagnosis: Listing of the possible diseases or conditions that may be responsible for the patient’s clinical presentation.

DISCUSSION

Characteristics of Bacillus Species

Bacillus species are large, motile, facultative anaerobic, gram-positive rods with a central spore. The spore is quite resistant to extreme conditions and can survive in nature for prolonged periods of time. Bacillus anthracis is nonmotile and on Gram stain is often seen in chains. The virulent forms of B. anthracis are more likely to be surrounded by a capsule. The organism can be cultured as large colonies on blood agar plates within 24 hours, often resembling a “Medusa head” (irregular appearance to the colony with swirling projections). The principal virulence factors of B. anthracis are the capsular polypeptide and anthrax toxin. The capsule consists of poly-D-glutamic acid, which is thought to allow the organism to resist phagocytosis. Anthrax toxin consists of three proteins: protective antigen, edema factor, and lethal factor. Protective antigen is named for its ability to confer immunity in experimental situations. Edema factor and lethal factor bind to protective antigen to form edema toxin and lethal toxin. The bound proteins are transported across cell membranes and are released in the cytoplasm where they exert their effects. Once the spores enter the body they are taken up by macrophages. Because of both lethal and edema factors, the spores survive killing, and subsequently germinate.

Diagnosis

The differential diagnosis of a patient (farmer) with fever, adenopathy, and black eschar include other cutaneous lesions such as furuncles (staphylococci), ecthyma gangrenosum (Pseudomonas aeruginosa), and spider bites. However, none of these etiologies are known to cause eschar formation with surrounding edema. The specific diagnosis of anthrax is made by growth of the organism from blood (inhalation anthrax), or wound (cutaneous anthrax).

Bacillus anthracis grows easily on most bacteriological culture media within 18–24 hours at 35°C (95°F). The organism is a nonmotile, spore-forming grampositive bacillus that is nonhemolytic when grown on blood containing agar medium and produces lecithinase on egg yolk agar. Lecithinase is an enzyme produced by both B. anthracis and B. cereus that degrades the lecithin in the

egg yolk agar leaving a white precipitate.

Careful review of a Gram stain from a primary specimen of a patient with suspected anthrax is necessary, because the organisms have the propensity to easily decolorize and appear gram-negative. However, the presence of spores is a key to the identification of the organism as a gram-positive bacillus. Based on these few tests (large gram-positive bacilli, nonhemolytic, lecithinase positive) a presumptive identification of B. anthracis can be made. As a result of the recent events in the world leading to concerns over bioterrorism, definitive diagnosis of anthrax must be performed in a public health laboratory. Confirmatory testing involves the use of fluorescently labeled monoclonal antibodies as well as DNA amplification assays. The use of India ink can also help to determine the presence of a capsule, a relatively unique aspect. The capsule of B. anthracis is not stained by the India ink, which can be easily visualized against the dark background.

Treatment and Prevention

Ciprofloxacin is the drug of choice for anthrax, following the identification of weaponized strains that were resistant to penicillin as a result of the production of a β-lactamase. Prevention of anthrax involves vaccination of animals as well as humans at high risk of exposure (military personnel). Prophylaxis is not recommended for asymptomatic persons. When deemed necessary, prophylaxis with ciprofloxacin must be maintained for up to 30 days because of the potential delay in germination of inhaled spores.

COMPREHENSION QUESTIONS

[1.1] A wound specimen obtained from a person working with wool from a Caribbean island demonstrated a large gram-positive rod from a nonhemolytic colony with swirling projections on blood agar. The most likely method to demonstrate spores would be which of the following?

A. Acid-fast stain

B. Gram stain

C. India ink stain

D. Malachite green stain

[1.2] Which of the following is the current preferred antimicrobial treatment of cutaneous anthrax?

A. Aminoglycosides

B. Ciprofloxacin

C. Penicillin

D. Tetracyclines

[1.3] Bacillus anthracis is unique to other bacteria. It is the only bacteria to possess which of the following?

A. An endotoxin

B. An exotoxin

C. A polypeptide capsule

D. A polysaccharide capsule

E. Lipopolysaccharide in its outer cell wall

F. Teichoic acid in its outer cell wall

Answers

[1.1] D. Spores can be observed as intracellular refractile bodies in unstained cell suspensions. Also, they are commonly observed by staining with malachite green or carbolfuchsin. The spore wall is relatively impermeable, but heating of the preparation allows dyes to penetrate. Alcohol treatment then serves to prevent spore decolorization. Finally, the spores are counterstained.

[1.2] B. Penicillin G was considered to be the first choice treatment for patients with cutaneous anthrax and when used should be continued for 7–10 days. However, because some naturally occurring isolates have been reported to be penicillin resistant (but still ciprofloxacin sensitive) and some patients are allergic to penicillin, ciprofloxacin is now considered to be the drug of choice for cutaneous anthrax. Ciprofloxacin belongs to the family of quinolones. As a fluorinated quinolone, it has greater antibacterial activity, lower toxicity, and is able to achieve clinically useful levels in blood and tissues compared to nonfluorinated quinolones. They act against many gram-positive and gram-negative bacteria by inhibiting bacterial DNA synthesis via the blockage of DNA gyrase. Despite the use of antibiotics in the treatment of anthrax, clinically manifested inhalational anthrax is usually fatal. If anthrax is suspected, public health authorities should be notified immediately. Aminoglycosides and tetracyclines have different mechanisms of action and have preferred uses in other disease states and infections. Aminoglycosides inhibit bacterial protein synthesis by attaching to and inhibiting the function of the 30S subunit of the bacterial ribosome. Their clinical usefulness has declined with the advent of cephalosporins and quinolones. Tetracyclines also inhibit bacterial protein synthesis; however they do so by inhibiting the binding of aminoacyl-tRNA to the 30S subunit of bacterial ribosomes.

[1.3] C. Virulent forms of B. anthracis, the causative agent of anthrax, are more likely to be surrounded by a capsule. This capsule is a polypeptide, composed of a polymer of glutamic acid, and is a unique feature of B. anthracis. Lipopolysaccharides (LPS/endotoxin) are unique to gram-negative bacteria (B. anthracis is a gram-positive rod). In addition, whereas B. anthracis is associated with both teichoic acid (cell wall) and a potent exotoxin, these are not unique features of this bacterium. Other gram-positives (i.e., staphylococci) release exotoxins and have teichoic acid in their cell walls.

MICROBIOLOGY PEARLS ❖ The most common form of anthrax is cutaneous anthrax in which penetration of the skin by B. anthracis spores causes eschar formation with regional lymphadenopathy. ❖ The organism is a nonmotile, spore-forming gram-positive bacilli that is nonhemolytic and produces lecithinase. ❖ Inhalation anthrax is a matter of public health concern. ❖ The drug of choice for treating anthrax is ciprofloxacin. ❖ The two main methods of anthrax virulence are its capsule and toxin. ❖ An eschar surrounded by edema is suspicious for anthrax.

REFERENCES

Logan NA, Turnbull PCB. Bacillus and other aerobic endospore-forming bacteria. In: 

Murray PR, Baron EJ, Jorgensen JH, et al., eds. Manual of Clinical Microbiology, 8th ed. Washington, DC: ASM Press 2003. Murray PR, Rosenthal KS, Pfaller MA. Bacillus. In: Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology, 5th ed. St. Louis, MO: Mosby, 2005:265–71. 

Ryan KJ. Sherris Medical Microbiology: An Introduction to Infectious Diseases, 3rd ed. New York: McGraw-Hill, 2003. 

Swartz MN. Recognition and management of anthrax—an update. N Engl J Med 2001;345:1621–6.

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