Agent and Pathogenesis

Last updated May 25, 2011

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Agent

Key microbiological characteristics of Bacillus anthracis follow (CDC 2001: Basic laboratory protocols for the presumptive identification of Bacillus anthracis, Martin 2010: Bacillus anthracis [anthrax]):

Vegetative cell: large, gram-positive bacillus (1.0 to 1.5 mcm x 3.0 to 5.0 mcm); "jointed bamboo-rod" appearance
Endospore: oval, central-to-subterminal, does not usually swell (1.0 x 1.5 mcm); CO₂ levels within the body inhibit sporulation
Forms long chains of vegetative cells in vitro; single cells or short chains of two to four cells in direct clinical samples
Readily forms spores in the presence of oxygen
Aerobic or facultatively anaerobic
Nonmotile
Catalase-positive
Nonhemolytic growth on sheep blood agar (SBA)
Rapid growth on SBA; comma-shaped projections may give "Medusa head" or "comet tail" appearance on SBA
Colonies have been described as having a "ground glass" or "curled hair" appearance and have the consistency of beaten egg whites
Colonies are 2 to 5 mm in diameter after 16 to 18 hours of incubation
Forms mucoid capsule when grown on agar with sodium bicarbonate and incubated in CO₂-enriched atmosphere; capsule can be visualized with India ink preparation
Susceptible to lysis by gamma phage

Spores germinate and form vegetative cells in environments rich in nutrients (eg, glucose, amino acids, nucleosides). Vegetative bacteria generally survive poorly outside of mammalian hosts. Conversely, vegetative cells form spores when nutrients in the environment are exhausted. Spores are protected by a morphologically complex protein coat (Giorno 2007). The exosporium may restrict dispersal and thereby increase the probability of a lethal dose for the grazing animal (Hugh-Jones 2009). Spores have been shown to have heat-resistance characteristics similar to other Bacillus species, and can survive in the environment for more than 40 years (Manchee 1990, Montville 2005).

Pathogenesis

Virulence Factors

The primary virulence factors produced by B anthracis are plasmid coded and include:

A poly-D-glutamic acid capsule (coded for on plasmid pXO2) that inhibits phagocytosis of vegetative bacilli
Three exotoxins that combine to produce two binary toxins:

Protective antigen (PA) is a binding protein that permits entry of toxin into host cells via endocyte formation. Once in the endocyte, PA toxin forms a pore, which creates a small passageway in the endosomal membrane that allows the enzymatic components of the toxin to enter the cytoplasm.
Edema factor (EF) is a calmodulin-dependent adenylate cyclase. EF combines with PA to form edema toxin (coded by the cya gene of plasmid pXO1).

Edema toxin converts adenosine triphosphate to cyclic adenosine monophosphate (cAMP); high intracellular levels of cAMP lead to impaired maintenance of water homeostasis and characteristic edema (Kumar 2002).
Edema toxin also inhibits neutrophil function and stimulates production or release of multiple inflammatory mediators, including neurokinins, prostanoids, and histamine (Tessier 2007).

Lethal factor (LF) is a zinc metalloprotease. LF combines with PA to form lethal toxin (coded by the lef gene on pXO1).

Lethal toxin is thought to stimulate overproduction of cytokines (eg, tumor necrosis factor [TNF] alpha and interleukin [IL] 1-beta), which leads to lysis of macrophages. Rapid release of inflammatory mediators also may contribute to the sudden death that can occur with anthrax.
Lethal toxin has been shown to cause endothelial cell apoptosis and endothelial barrier dysfunction, which may contribute to vascular destruction (Kirby 2004, Warfel 2005).

Virulence of B anthracis appears to be related to clonality and to the numbers of copies of the pXO1 and pXO2 plasmids within each bacterial cell (Coker 2003). The complete genomic sequence of B anthracis (Ames strain) has been analyzed; several chromosomally encoded potential virulence factors were identified, including hemolysin, phospholipases, and iron acquisition proteins (Read 2003).

Anthrax toxin genes were identified in a naturally occurring B cereus isolate obtained from a patient with an illness similar to inhalational anthrax (Hoffmaster 2004). More recently, anthrax-like illnesses were identified in two metalworkers who had inhalation exposure to B cereus that contained B anthracis toxin genes (Avashia 2007).

Cutaneous Anthrax

The pathogenesis of cutaneous anthrax involves the following process:

Endospores are introduced through the skin (usually via preexisting skin lesions or abrasions).
Low-level germination at the site of introduction produces localized necrosis with eschar formation and soft-tissue or mucosal edema (which can be massive in some cases). Epithelial damage appears to be required for germination of spores. Germination begins 1 to 3 hours after inoculation, but spore germination by itself is not sufficient to produce infection in undamaged skin (Bischof 2007).
Endospores often are phagocytosed by macrophages and carried to regional lymph nodes, causing painful lymphadenopathy and lymphangitis.
Hematogenous spread with resultant toxemia can occur, although such spread is not common with appropriate antibiotic therapy.
The infective dose for cutaneous anthrax is not known.

Inhalational Anthrax

Pathogenesis of inhalational anthrax involves the following steps (Abramova 1993, Hanna 1998):

Endospores are introduced into the body via inhalation. Endospores are 1 mcm x 1.5 mcm in size (CDC 2001: Basic laboratory protocols for the presumptive identification of Bacillus anthracis) and are, therefore, able to reach the alveoli (ie, <5 mcm).
Endospores are phagocytosed by macrophages and carried to regional lymph nodes. They also appear to be taken up by lung epithelial cells (Russell 2008).
The endospores then germinate inside macrophages and become vegetative cells, which leave the macrophages and multiply in the lymphatic system.
Bacteria enter the bloodstream and lead to septic shock and toxemia; hematogenous spread can lead to hemorrhagic meningitis.
Regional hemorrhagic lymphadenitis of mediastinal and peribronchial lymph nodes causes hemorrhagic mediastinitis (Abramova 1993). A widened mediastinum may be noted on chest radiograph or enlarged lymph nodes may be directly visualized on chest computed tomography.
Pulmonary lymphatic drainage can be blocked, leading to pulmonary edema.
Pleural effusions are common and may be massive. B anthracis bacilli, bacillary fragments, and antigens can be noted with immunohistochemistry (IHC) testing of pleural effusions (Guarner 2003).
True pneumonia rarely occurs, although a focal, hemorrhagic, necrotizing pneumonic lesion (similar to the Gohn complex of tuberculosis) may be seen (Abramova 1993). Intraalveolar edema, focal areas of hyaline membrane formation, and interstitial mononuclear inflammation may be noted (Guarner 2003).
Compression of the lungs and septic shock are the major causes of death.
The median infective dose (ID₅₀) for inhalational anthrax is estimated at 8,000 to 50,000 spores (Franz 1997), although the minimum infective dose may be considerably less. On the basis of experimental studies involving primates, the US Department of Defense (DoD) has estimated that the median lethal dose (LD₅₀) for inhalational anthrax in humans from weapons-grade anthrax is 2,500 to 55,000 spores (DIA 1986).
Extrapolation of dose-response curves involving cynomolgous monkeys suggest that the LD₁₀ (dose at which 10% of the population is expected to die) in humans following exposure to airborne anthrax spores may be as low as 50 to 98 spores, the LD₅ (5% fatalities) may be only 14 to 28 spores, and the LD₁ (1% fatalities) may be only 1 to 3 spores (Peters 2002).
Mathematical modeling of airborne anthrax infection based on observations from the US Postal Service experience during the 2001 anthrax outbreak (and an assumption that 10,000 people may have been exposed) suggests that exposures ranged from 18 to 863 spores and may have been as low as 2 to 9 spores (Fennelly 2004).
The dose-response relationship for inhaled B anthracis is highly uncertain. However, a review of animal and human studies, including those that documented spore exposures insufficient to cause inhalational anthrax, suggests that an exposure threshold may exist (Coleman 2008).
Animal data have suggested that anthrax spores might be able to survive in the lungs for longer than 60 days; in one study, live spores were detected in the lymph nodes of a monkey 100 days after exposure (Henderson 1956).

Gastrointestional Anthrax

The pathogenesis of gastrointestinal anthrax is not clear, since this condition is relatively rare. Historically, illness has been thought to result from ingestion of B anthracis spores; however, recently experts have postulated that illness predominantly results from ingestion of large numbers of vegetative cells (such as may be found in poorly cooked meat from infected animals) (Inglesby 2002).

Two forms of gastrointestinal anthrax have been recognized: oropharyngeal and abdominal.
In oropharyngeal anthrax, the portal of entry is the oral or pharyngeal mucosa. A mucosal ulcer occurs initially, followed by regional lymphadenopathy and localized edema.
In abdominal anthrax, the portal of entry often is the terminal ileum or cecum. Intestinal lesions occur and are followed by regional lymphadenopathy. Edema of the bowel wall and ascites (sometimes massive) may be present.
Hematogenous spread with resultant toxemia can occur.
The infective dose for gastrointestinal anthrax is not known. In animal models (guinea pigs, rabbits, and rhesus monkeys), investigators failed to induce infection following oral challenge with 10⁸ spores; these animals are all thought to be more susceptible to anthrax than humans (Beatty 2003).


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