Microbiology, pathogenesis, and epidemiology of anthrax

INTRODUCTION — Bacillus anthracis, which causes anthrax, was the first clearly recognized bacterial pathogen. The life cycle of the organism was unraveled by Koch, who recognized the importance of dormant anthrax spores in the perpetuation of the organism in soil. These studies eventually helped to underpin Koch's postulates, a milestone in establishing specific pathogens as the causative agents of human and animal diseases. Pasteur created the first successful antibacterial vaccine by successfully attenuating strains of B. anthracis and then proving that these strains could protect sheep from infection with fully virulent strains.

The microbiology, pathogenesis, and epidemiology of anthrax will be reviewed here. The clinical manifestations, diagnosis, treatment, and prevention of anthrax are discussed separately. (See "Clinical manifestations and diagnosis of anthrax" and "Treatment of anthrax" and "Prevention of anthrax".)

MICROBIOLOGY — B. anthracis is a sporulating gram-positive rod (picture 1). It is nonmotile and grows rapidly at 37ºC on blood agar plates under aerobic conditions. Individual colonies are nonhemolytic and sticky. B. anthracis is not phylogenetically distinct from Bacillus cereus and is considered a clade within the species B. cereus [1]. However, because of its medical significance, B. anthracis retains its original species name.

Molecular techniques can be used to confirm the identity of the organism, distinguishing it from Bacillus thuringiensis, and polymerase chain reaction techniques are highly sensitive for detecting B. anthracis. However, distinguishing B. anthracis and other clades of B. cereus can be difficult [2]. Mass spectrometry appears to be a reliable method [3,4]. Additionally, anthrax syndromes caused by strains of other members of the B. cereus complex that carry the pXO1 virulence plasmid have been reported [5,6].

Thus, if a B. cereus clade organism is isolated from a patient with a disease suggestive of anthrax, the patient should be treated for anthrax and the isolate sent to a reference lab.

There is less genomic variation in B. anthracis than in any other known bacterial species that cause disease in humans. As a result, strain differentiation relies on finding single-nucleotide polymorphisms by whole-genome sequencing [7], though more focused sequencing appears promising [8].

PATHOGENESIS

Portals of entry and dissemination — B. anthracis can invade the body by four routes: ingestion, transcutaneous inoculation, inhalation, and direct parenteral injection.

●Gastrointestinal tract – Although disseminated anthrax frequently seeds the gastrointestinal tract [9], the term "gastrointestinal anthrax" is used to refer to cases in which the organism gains access through ingestion. Most anthrax in nature occurs in herbivores and is acquired via the gastrointestinal route. Spores contaminating grasses are ingested. The organism then invades gut-associated lymphoid tissue, especially at Peyer's patches. An in vitro study using a cell line derived from human M cells suggests that, as in several other infections, the M cell plays a role in bacterial cell uptake [10]. In humans, gastrointestinal anthrax follows ingestion of contaminated and undercooked meat. Following ingestion, anthrax bacilli are transported to mesenteric lymph nodes. Subsequently, hemorrhagic mesenteric adenitis, ascites, and septicemia may occur. One case was described in a patient who had played a drum made with a contaminated animal skin. An investigation suggested that the patient was exposed to B. anthracis spores at the drumming event, which resulted in gastrointestinal anthrax rather than inhalation anthrax [11]. (See 'Animal hides' below.)

●Skin – Invasion through the skin may occur via two mechanisms. Although skin abrasions were thought to be necessary for cutaneous infection, the organism also appears to be able to invade hair follicles [12]. Once B. anthracis spores introduced under the skin become vegetative organisms and begin to multiply, production of an antiphagocytic capsule facilitates local spread, and exotoxin production produces marked edema and tissue necrosis. An eschar with extensive surrounding edema is the hallmark of cutaneous anthrax.

●Inhalation – The classic understanding of inhalation anthrax is that spores must reach the terminal bronchioles and alveoli, where they spread to the mediastinal lymph nodes by a process that appears to involve dendritic cells [13]. There, they germinate and induce a hemorrhagic mediastinitis before disseminating. Less frequently, inhaled spores can cause nasal, pharyngeal, or laryngeal disease or patients may present with primary meningitis, where the portal of entry is most likely the respiratory tract [14]. Airborne anthrax spores >5 microns in size pose no threat to the lungs, since they are either physically trapped in the nasopharynx or cleared by the mucociliary escalator system [15]. Spores between 1 and 5 microns in size are deposited in alveolar ducts or alveoli. These spores are phagocytosed by alveolar macrophages and transported to mediastinal lymph nodes, where they multiply and cause hemorrhagic mediastinitis [9,16,17]. Bacteremia is a near-universal complication of untreated mediastinal infection. Larger particles may lodge in the upper respiratory tract and cause infection, which can disseminate from that site [18].

●Direct injection – Direct injection into the host can occur when contaminated heroin is used [19]. This has occurred by the transcutaneous route and intravenously when heroin is taken parenterally. (See 'Injection anthrax' below.)

Whatever the portal of entry, infection can lead to disseminated disease; if not treated early, inhalation anthrax does so in all cases. Sites of infection such as the mediastinum, the meninges, and the intestines show hemorrhagic inflammation [20].

The clinical manifestations of anthrax are discussed in greater detail separately. (See "Clinical manifestations and diagnosis of anthrax", section on 'Clinical manifestations'.)

Virulence factors — Virulent B. anthracis requires a poly-D-glutamic acid capsule and three proteins (edema factor [EF], lethal factor [LF], and protective antigen [PA]) [21]. Virulence is enhanced by the cell surface protein BslA, which mediates adhesion to host cells [22-24]. Other virulence factors have been suggested, but induction of specific pathological events by these other factors has not been established. The organism changes its metabolism in several ways in the in vitro environment [25].

Toxin and capsule production are dependent upon the presence of two plasmids:

●pX01 (184.5 kbp) is required for the production of the components of the two exotoxins.

●pX02 (95.3 kbp) contains the genes for synthesis of the poly-D-glutamic acid capsule.

The morbidity and mortality associated with anthrax requires the production of two toxins formed by three components: PA (protective antigen), EF (edema factor), and LF (lethal factor). EF and LF each combine with PA to form the two binary exotoxins, edema toxin (ET) and lethal toxin (LT), respectively [26]. The PA, EF, and LF genes are under the control of the anthrax toxin activator gene (AtxA), which is encoded on pX01. The increased CO₂ and HCO₃- concentrations of the host environment increase AtxA activity [27]. The toxins interfere with the host response early in the infection and lead to mortality late in the infection when the bacteria have reached high concentrations in the bloodstream [21]. The poly-D-glutamic acid capsule is antiphagocytic and weakly antigenic. Thus, strains without the pX02 plasmid are unencapsulated and are avidly phagocytosed by polymorphonuclear leukocytes [28]. Capsule synthesis is under the indirect control of AtxA, which induces regulators that in turn control transcription of capsular synthetic genes. AtxA also regulates a variety of chromosomal genes that may form a regulatory network involved in pathogenesis [29].

Protective antigen — Protective antigen (PA) is antigenic and antibodies to it are highly protective against anthrax infection [30]. It is the binding moiety for the two toxins and attaches to the host surface proteins ANTXR1 (tumor endothelial marker 8) and the similar ANTXR2 [30,31]. Heterogeneity of ANTXR2 may determine different degrees of disease susceptibility [32]. Upon attaching to the cell surface, PA is cleaved, allowing for two functions: polymerization into heptamers that form a cation-selective channel in the cell membrane and binding of LF or EF, which enter the cytosol through the channels [33-35]. Synthetic fusion of other proteins to the N-terminal domain of LF allows them to enter the cytosol as well [36]; this mechanism is being investigated as a means of delivering a variety of therapeutic agents to the cytosol [37,38].

Lethal factor and edema factor — LF is a zinc metallo-protease that cleaves mitogen-activated protein (MAP) kinases 1 and 2, leading to their inactivation and inhibition of the MAP kinase signal transduction pathway [39]. This inhibition of the MAP kinase pathway leads to inhibition of upstream signaling components that mediate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase assembly.

EF is a calcium-bound calmodulin-stabilized toxin that efficiently converts adenosine triphosphate (ATP) to cyclic adenosine monophosphate (cAMP) [40,41]. The accumulation of cAMP leads to activation of the kinase A signaling pathway and thus to activation of many cellular functions.

Early in infection, LF and EF interfere with innate immune functions, allowing the organism to multiply and disseminate [21]. Early on, neutrophils, macrophages, and dendritic cells are affected. In neutrophils, recruitment, chemotaxis, priming, phagocytosis, cytokine production, and superoxide production are suppressed [42-46]. The toxins inhibit recruitment by macrophages and mechanisms by which macrophages kill bacteria [47,48]. Macrophages and dendritic cells succumb to activation of caspase 1 and programmed cell death [49]. A study of organ-specific and whole-body metabolomics in mice also reflected alterations in lipid metabolism associated with dysfunction of innate immunity [50]. As a result of effects on innate immunity, B. anthracis multiplies to high concentrations in lymph nodes and blood with a likely lethal outcome if this stage is reached. As an example, during the 12-hour period preceding death of guinea pigs infected with anthrax bacilli, the number of bacteria in the blood rose from 300,000 to one billion organisms/mL [51].

In late disseminated anthrax, the toxins are widely active at inaccessible intracellular locations and treatment does not assure a good outcome. Toxin levels in blood are also very high during this stage. In guinea pigs, if antibiotics are given after intravascular bacterial concentrations reach one million organisms/mL, the animals still die despite a marked reduction in these concentrations [51]. Furthermore, sterile blood from these guinea pigs reproduces a fatal toxemic syndrome when given to normal guinea pigs. Both ET and LT lead to vascular collapse in experimental animals [52,53]. ET induces multiorgan hemorrhage [53]. ET and LT are toxic to essentially all cell types and study of host metabolomics indicates that many metabolic pathways are affected in vivo even early in the disease [50], but studies in PA-receptor knockout mice have indicated which organs are the most damaged at least in mice. Intuitively, it might seem that given the hypotension and edema seen in anthrax, vascular endothelium would be a major type of tissue to fail, but this does not appear to be the case [54]. In mice, myocardium and smooth muscle are the primary targets of LT [55,56], but LT also induces lung injury [57]. ET is less well studied in this model. It is thought to have widespread effects, but one study suggests that ET-induced lethality occurs via its effects on hepatocytes [58].

Immune response — The immune response to high-level anthrax exposure was evaluated in persons exposed or possibly exposed to anthrax when a letter containing anthrax spores was sent to the Senate Office Building in the United States in 2001 [59,60]. All of these individuals were immediately treated with antibiotics and none developed clinical anthrax, but postexposure antibiotic prophylaxis did not prevent stimulation of the immune system [59]. Antibodies to PA and LF were present in approximately 40 and 14 percent of unvaccinated individuals, respectively, and evidence of cell-mediated immunity to PA and LF was present in about 80 and 60 percent, respectively. Although the immune responses were generally of low magnitude, there was a dose-response gradient, with immune responses primarily occurring in individuals with higher levels of exposure. (See 'Bioterrorism' below.)

The presence of LF in the serum and immune responses were evaluated in 26 patients who developed cutaneous anthrax during an outbreak in Bangladesh in 2009 [61]. LF was detected in acute serum samples from 18 of 26 individuals. Anti-PA immunoglobulin (Ig)G and lethal toxin neutralizing antibodies were detected in the sera from these 18 individuals. Seroconversion to serum anti-PA IgG and/or lethal toxin neutralizing antibodies occurred only in patients with measurable LF in the serum. Nine of the 18 patients who had LF in the serum and anti-PA IgG had received antimicrobial therapy two to seven days prior to the first sample collection, suggesting that antimicrobial use does not interfere with the development of a humoral immune response.

EPIDEMIOLOGY — The organism is predominantly a pathogen of mammals, but it can survive and sometimes multiply in soil [62,63]. Despite the rarity of human cases in the current era, anthrax remains a potential threat for two reasons: anthrax epizootics still occur, and the organism remains an important potential agent of bioterrorism and biological warfare.

Epizootic anthrax — B. anthracis can be isolated from soil at sites where anthrax-infected animal carcasses have been, and, when conditions are favorable, it may propagate in soil [63]. However, the organism is considered to be predominantly a pathogen of grazing animals; the extent to which multiplication in soil plays a role in its epidemiology is not clear.

Spores can persist in the soil for decades. Surface decontamination is not practical except in unusual circumstances; thus, epizootic anthrax (ie, outbreaks in animals) will continue to occur in highly endemic areas in the developing world, where the extent of the use of animal anthrax vaccine is suboptimal [64-67]. B. anthracis is also endemic in agricultural regions of South and Central America, central and southwestern Asia, Africa, and southern and eastern Europe [68].

Systemic anthrax is primarily a disease of herbivores. Humans become accidentally infected through contact with infected animals, their carcasses, or their products. In the 1950s and 1960s, over 80 percent of cases in the United States were related to products that were manufactured from imported goat hair [69]. Inhalation anthrax, or wool-sorters' disease, follows the inhalation of anthrax spores generated during the early cleaning of contaminated goat hair.

During the 20^th and 21^st centuries, improvements in industrial hygiene, a decrease in the use of imported, contaminated animal materials, and immunization of at-risk workers resulted in a reduction in the United States incidence of inhalation anthrax (only 18 cases in the United States) [17]. Before the 2001 bioterrorism attack, the last prior fatal case of anthrax in the United States occurred in 1976 when a weaver died of inhalational anthrax after working with yarn imported from Pakistan [70].

Epizootic anthrax cases occur rarely in the United States. In 2000, 32 farms in North Dakota in the United States were quarantined because of anthrax: a total of 157 animals died during this epizootic, and a single ranch worker who helped move dead animals developed cutaneous anthrax [71].

Animal hides — Although the risk of anthrax associated with the handling of animal hides is low, such cases still sporadically occur [11,72,73]. As an example, a man in Connecticut in the United States developed cutaneous anthrax in 2007 after processing a contaminated African goat hide to make a traditional drum [73]. His eight-year-old child also developed cutaneous anthrax despite having had no direct contact with the hide. An investigation revealed widespread contamination of multiple areas of the home with B. anthracis, although all drum-making activities were confined to a backyard shed. In 2009, a 24-year-old woman in New Hampshire in the United States developed gastrointestinal anthrax after briefly playing a contaminated animal-hide drum [11,74].Environmental sampling showed contamination of the drum and the environment.

Injection anthrax — In 2000, systemic anthrax was reported in a heroin user in Norway [19]. There was a time gap, then an outbreak of anthrax occurred in Scotland spreading to England and Germany between December 2009 and December 2010 affecting 119 heroin users; there were 47 confirmed cases, 35 probable cases, and 37 possible cases, with 14 deaths [75,76]. In 2012 to 2013, there were 70 cases with 26 fatalities in Denmark, France, Germany, and the United Kingdom [77], and cases have continued to occur sporadically [78]. Whole-genome sequencing of 60 isolates has shown multiple strains forming two closely related groups [79]. One group was associated with the 2009 to 2010 outbreak and a different group with the 2012 to 2013 outbreak. Intriguingly, the original isolate from Norway belonged to the second group. Molecular epidemiology has made it possible to determine the geographic origin of B. anthracis isolates, and both clades appear to originate in the Middle East, possibly Turkey. The clinical features are discussed in greater detail separately.

Bioterrorism — In 2001 in the United States, 22 cases of anthrax, 18 confirmed and 4 suspected, resulted from attempts to deliberately expose selected individuals or organizations to weaponized anthrax spores [80-84]. Eleven of these cases were inhalational and 11 cutaneous; all but two inhalational cases resulted from exposure to B. anthracis in a powder that had been sent through the mail, and most of the inhalational cases occurred in postal employees [85]. Many unaffected people developed symptoms [86], and the event was estimated to have cost the health care system USD $177 million, largely for care of people with no history of exposure [87].

Two unexpected findings resulted from the investigations of these bioterrorism cases. First, airborne dissemination of anthrax spores occurred from sealed envelopes during their travel through high-speed mail sorting machines. Second, re-aerosolization of infective spores occurred long after airborne spores had settled onto surfaces.

Twenty-five days after the Senate Office Building was closed, a study was conducted in the office of a United States senator who had received an envelope that was opened by his staff [88]. Individuals wearing sterile protective suits initially placed sampling devices around the office suite and then left the area. Later they returned to the contaminated areas and simulated office activity such as walking, sorting mail, and moving trash cans. Airborne spore concentrations increased 65-fold during the simulated active period, proving that re-aerosolization of anthrax spores is possible.

Analysis of variable-number tandem repeat segments of DNA indicated that the Ames strain had been used in these attacks. This strain had been used widely by the United States military in biodefense research and distributed minimally to nonmilitary laboratories. Civilian laboratories were eliminated as the source by a criminal investigation. Comparing whole-genome single-nucleotide polymorphisms (SNPs) found in the spores sent in the mail with SNPs from laboratory samples, the United States Centers for Disease Control and Prevention (CDC) and the Federal Bureau of Investigation (FBI) implicated Bruce Ivins, a biodefense researcher working at the United States Army Medical Research Institute in Frederick, Maryland. The case was never submitted to the scrutiny of a court of law since the researcher committed suicide before the case could be tried [89]. In 2011, a panel of experts assembled by the National Academy of Sciences evaluated the genetic analysis of the anthrax spores and advised that the scientific evidence put forth by the FBI was insufficient to prove that Ivins was the culprit [90]. One anthrax authority and coworkers have pointed out that the spores contained large amounts of tin and silicon, probably used as a coating and possibly linking them to Dugway Proving Ground (a United States Army facility for testing biologic and chemical weapons), the source of Bruce Ivins strain in question [91].

Laboratory incidents — In March 2002, an unvaccinated laboratory worker in Texas in the United States developed cutaneous anthrax after handling agar plate cultures and anthrax-containing vials of environmental samples of B. anthracis as part of the investigation of the bioterrorist attack of 2001 [92]. Although B. anthracis is not a highly infectious organism in the laboratory, its potential severity makes it important to carefully follow biosafety recommendations.

Sverdlovsk accident — An anthrax epidemic that affected both humans and livestock occurred in Sverdlovsk in the former Soviet Union in 1979 due to accidental release from a military bioweapons facility [93]. At the time, Soviet authorities attributed the epidemic to consumption of contaminated meat. American and Russian scientists collaborated on an epidemiologic study reported in 1994, which showed the distribution of cases followed a plume that would have been explained by the prevailing wind at the time [93]. More recently, whole-genome sequencing of anthrax genome from autopsy specimens has indicated that the strain was from Asia and had not been genetically modified [94].

SUMMARY

●Bacillus anthracis, which causes anthrax, was the first clearly recognized bacterial pathogen. The life cycle of the organism was unraveled by Koch, who recognized the importance of dormant anthrax spores in the perpetuation of the organism in soil. (See 'Introduction' above.)

●B. anthracis is a sporulating gram-positive rod. Virulent B. anthracis requires a poly-D-glutamic acid capsule and three proteins (edema factor [EF], lethal factor [LF], and protective antigen [PA]) that associate into two protein exotoxins. (See 'Pathogenesis' above.)

●B. anthracis has the potential to invade the body by four routes: ingestion, transcutaneous inoculation, inhalation, and direct parenteral injection. Whatever the portal of entry, infection can lead to disseminated disease; if not treated early, inhalation anthrax does so in all cases. (See 'Portals of entry and dissemination' above.)

●Gastrointestinal anthrax follows ingestion of contaminated and undercooked meat. Following ingestion, anthrax bacilli are transported to mesenteric lymph nodes. Subsequently, hemorrhagic mesenteric adenitis, ascites, and septicemia may occur. (See 'Portals of entry and dissemination' above.)

●When introduced subcutaneously, spores of virulent B. anthracis become vegetative organisms and begin to multiply. Subsequent production of an antiphagocytic capsule facilitates local spread, and exotoxin production produces marked edema and tissue necrosis. An eschar with extensive surrounding edema is the hallmark of cutaneous anthrax. (See 'Portals of entry and dissemination' above.)

●Spores between 1 and 5 microns in size are deposited in alveolar ducts or alveoli. Larger groupings of spores are usually filtered out or trapped by the mucociliary tree and can cause infection of the nasal passage, pharynx, and larynx. Spores in the lung are phagocytosed by alveolar macrophages and transported to mediastinal lymph nodes, where they multiply and cause a hemorrhagic mediastinitis. Bacteremia is a near-universal complication after mediastinal infection has become established. (See 'Portals of entry and dissemination' above.)

●The organism's major ecologic niche is as a pathogen of herbivores. It also may be able to propagate in soil. Humans become accidentally infected through contact with infected animals or their products. (See 'Epidemiology' above.)

●In 2001 in the United States, 22 cases of anthrax, 18 confirmed and 4 suspected, resulted from attempts to deliberately expose selected individuals or organizations to weaponized anthrax spores. Eleven of these cases were inhalational and 11 cutaneous; all but two inhalational cases resulted from direct exposure to B. anthracis in a powder that had been sent through the mail, and most of the inhalational cases occurred in postal employees. (See 'Bioterrorism' above.)

●Large outbreaks of anthrax occurred in heroin users in Europe in 2009 to 2010 and 2012 to 2013, causing skin and soft tissue infections in most patients. Additional cases have been reported in heroin users in Europe subsequently. Two separate contamination events appear to have occurred. (See 'Injection anthrax' above.)