Bacillus anthracis and anthrax

Introduction

The anthrax bacillus, Bacillus anthracis, was the first bacterium shown to be the cause of
a disease. In 1877, Robert Koch grew the organism in pure culture, demonstrated its
ability to form endospores, and produced experimental anthrax by injecting it into
animals.

Figure 1. Robert Koch's original photomicrographs of Bacillus anthracis, the agent of anthrax.
Compare the cell morphology and spore position with the Gram stain below (Figure 2). This is
Bacillus anthracis. Beware of phony and mislabeled images of B. anthracis on the internet, including
some that are posted by otherwise credible websites. Look for large cells with square ends and
centrally-located ellipsoid spores when identifying Bacillus anthracis.

Bacillus anthracis is very large, Gram-positive, sporeforming rod, 1 - 1.2µm in width x 3

- 5µm in length. The bacterium can be cultivated in ordinary nutrient medium under
aerobic or anaerobic conditions. Genotypically and phenotypically it is very similar to
Bacillus cereus, which is found in soil habitats around the world, and to Bacillus
thuringiensis, the pathogen for larvae of Lepidoptera. The three species have the same
cellular size and morphology and form oval spores located centrally in a nonswollen
sporangium.

Figure 2. Bacillus anthracis. Gram stain. 1500X. The cells have characteristic squared ends. The
endospores are ellipsoidal shaped and located centrally in the sporangium. The spores are highly
refractile to light and resistant to staining.

Bacillus thuringiensis is distinguished from B. cereus or B. anthracis by its pathogenicity

for Lepidopteran insects (moths and caterpillars) and by production of an intracellular
parasporal crystal in association with spore formation. The bacteria and protein crystals
are sold as "Bt" insecticide, which is used for the biological control of certain garden and
crop pests.
Figure 3. Bacillus thuringiensis. Phase Photomicrograph of vegetative cells, intracellular spores (light)
and parasporal crystals (dark). 1000X.

Bacillus cereus is a normal inhabitant of the soil, but it can be regularly isolated from
foods such as grains and spices. B. cereus causes two types of food-borne intoxications
(as opposed to infections). One type is characterized by nausea and vomiting and
abdominal cramps and has an incubation period of 1 to 6 hours. It resembles
Staphylococcus aureus food poisoning in its symptoms and incubation period. This is the
"short-incubation" or emetic form of the disease. The second type is manifested primarily
by abdominal cramps and diarrhea with an incubation period of 8 to 16 hours. Diarrhea
may be a small volume or profuse and watery. This type is referred to as the "long-
incubation" or diarrheal form of the disease, and it resembles food poisoning caused by
Clostridium perfringens. In either type, the illness usually lasts less than 24 hours after
onset.

The short-incubation form is caused by a preformed, heat-stable emetic toxin, ETE. The
mechanism and site of action of this toxin are unknown, although the small molecule
forms ion channels and holes in membranes. The long-incubation form of illness is
mediated by the heat-labile diarrheagenic enterotoxin Nhe and/or hemolytic enterotoxin
HBL, which cause intestinal fluid secretion, probably by several mechanisms, including
pore formation and activation of adenylate cyclase enzymes.

Figure 4. Bacillus cereus. Gram stain. 450X. Bacilli are large bacteria, so that they are readily
observed with the microscope's "high dry objective" ........but you can't detect anything about their
spores. This could be a Lactobacillus.

Cultivation

Several nonselective and selective media for the detection and isolation of Bacillus
anthracis have been described, as well as a rapid screening test for the bacterium based
on the morphology of microcolonies. Table 1 provides the differential characteristics that
are used to distinguish Bacillus anthracis from most strains of Bacillus cereus and
Bacillus thuringiensis but not necessarily from other saprophytic species of Bacillus.
Otherwise, it is not the intent of this article to provide information on the growth of the
bacterium in the laboratory.
 Table 1. Differential Characteristics of B. anthracis B. cereus and B. thuringiensis

Characteristic
growth requirement for thiamin
hemolysis on sheep blood agar
glutamyl-polypeptide capsule
lysis by gamma phage
motility
growth on chloral hydrate agar
string-of-pearls test
B. cereus and
B. anthracis
B. thuringiensis
+ -
- +
+ -
+ -
- +
- +
+ -

The following figures (5, 6, and 7) from the CDC are reliable images of Bacillus anthracis grown as
described in the figure legends.

Figure 5. Colonies of Bacillus cereus on the left; colonies of Bacillus anthracis on the right. B. cereus
colonies are larger, more mucoid, and this strain exhibits a slight zone of hemolysis on blood agar.

Figure 6. Lysis of Bacillus anthracis by the lytic phage gamma. The plaque (clear area) in the region of
confluent growth is where the gamma phage was applied. The plaque results from the phage's ability to
lyse the bacterial cells. Since the gamma phage is specific for B. anthracis, and will not lyse B.
thuringiensis or B. cereus, we know that this is Bacillus anthracis. The colony type of is similar to Figure
5.
Figure 7. Mucoid colonies of Bacillus anthracis. This culture was probably incubated at an increased
CO2 tension (5% CO2) which greatly enhances production of the poly-D-glutamyl capsule and
accounts for the mucoid colony type.

Anthrax

Anthrax is primarily a disease of domesticated and wild animals, particularly herbivorous

animals, such as cattle, sheep, horses, mules and goats. Humans become infected
incidentally when brought into contact with diseased animals, which includes their flesh,
bones, hides, hair and excrement.

The natural history of Bacillus anthracis is obscure. Although the spores have been found
naturally in soil samples from around the world, the organisms cannot be regularly
cultivated from soils where there is an absence of endemic anthrax. In the United States
there are recognized areas of infection in South Dakota, Nebraska, Arkansas, Texas,
Louisiana, Mississippi, California and small areas that exist in other states. Even in
endemic areas, anthrax occurs irregularly, often with many years between occurrences.

In the United States, the incidence of naturally-acquired anthrax is extremely rare (1-2
cases of cutaneous disease per year). Worldwide, the incidence is unknown, although B.
anthracis is present in most of the world. Unreliable reporting makes it difficult to
estimate the true incidence of human anthrax worldwide. However, in fall 2001, 22 cases
of anthrax (11 inhalation, 11 cutaneous) were identified in the United States following
intentional contamination of the mail.

The most common form of the disease in humans is cutaneous anthrax, which is usually
acquired via injured skin or mucous membranes. A minor scratch or abrasion, usually on
an exposed area of the face or neck or arms, is inoculated by spores from the soil or a
contaminated animal or carcass. The spores germinate, vegetative cells multiply, and a
characteristic gelatinous edema develops at the site. This develops into papule within
12-36 hours after infection. The papule changes rapidly to a vesicle, then a pustule
(malignant pustule), and finally into a necrotic ulcer from which infection may
disseminate, giving rise to septicemia. Lymphatic swelling also occurs within seven days.
In severe cases, where the blood stream is eventually invaded, the disease is frequently
fatal.

Another form of the disease, inhalation anthrax (woolsorters' disease), results most
commonly from inhalation of spore-containing dust where animal hair or hides are being
handled. The disease begins abruptly with high fever and chest pain. It progresses rapidly
to a systemic hemorrhagic pathology and is often fatal if treatment cannot stop the
invasive aspect of the infection.
Gastrointestinal anthrax is analogous to cutaneous anthrax but occurs on the intestinal
mucosa. As in cutaneous anthrax, the organisms probably invade the mucosa through a
preexisting lesion. The bacteria spread from the mucosal lesion to the lymphatic system.
Intestinal anthrax results from the ingestion of poorly cooked meat from infected animals.
Gastrointestinal anthrax is rare but may occur as explosive outbreaks associated with
ingestion of infected animals. Intestinal anthrax has an extremely high mortality rate.

Meningitis due to B. anthracis is a very rare complication that may result from a primary
infection elsewhere.

Pathogenicity of Bacillus anthracis

Bacillus anthracis clearly owes its pathogenicity to two major determinants of virulence:
the formation of a poly-D-glutamyl capsule, which mediates the invasive stage of the
infection, and the production of the multicomponent anthrax toxin which mediates the
toxigenic stage.

Poly-D-glutamyl capsule

Bacillus anthracis forms a single antigenic type of capsule consisting of a poly-D-

glutamate polypeptide. All virulent strains of B. anthracis form this capsule. Production
of capsular material is associated with the formation of a characteristic mucoid or
"smooth" colony type. "Smooth" (S) to "rough" (R) colonial variants occur, which is
correlated with ability to produce the capsule. R variants are relatively avirulent. Capsule
production depends on a 60 megadalton plasmid, pX02; its transfer to nonencapsulated
B. anthracis via transduction produces the encapsulated phenotype.

Figure 8. Two microscopic techniques to demonstrate the presence of the poly-D-glutamyl capsule of
Bacillus anthracis. Left. India ink capsule outline 1000X. Right a fluorescent-labeled antibody is
reacted specifically with the capsular material which renders the capsule fluorescent - FA stain
1000X.

The poly-D-glutamyl capsule is itself nontoxic, but functions to protect the organism
against complement and the bactericidal components of serum and phagocytes, and
against phagocytic engulfment and destruction. The capsule plays its most important role
during the establishment of the infection, and a less significant role in the terminal phases
of the disease, which are mediated by the anthrax toxin.

The poly-D-glutamyl capsule is formed in vivo or in the laboratory when the bacterium is
grown on serum plates in a 5% CO2 atmosphere. The capsular material can be detected by
the McFadyean reaction which involves staining with polychrome methylene blue. Blue
rods in a background of purple/pink-stained capsular material is a positive test (Figure 9).
Neither B. cereus nor B. thuringiensis synthesizes this capsular polymer, so the detection
of capsular material can be used to distinguish B. anthracis from its closest relatives.

Figure 9. McFadyean's reaction showing short chains of Bacillus anthracis cells lying among
amorphous, disintegrated capsular material. White blood cells can also be seen.

Anthrax Toxin

The toxigenic properties of Bacillus anthracis were not recognized until 1954. Prior to
that time, because of the tremendous number of anthrax bacilli observed in the blood of
animals dying of the disease (109 bacteria/ml), it was assumed that death was due to
blockage of the capillaries, popularly known as the "log-jam" theory. But experimentally
it was shown that only about 3 x 106 cells/ml are necessary to cause death of the animal.
Furthermore, the cell-free plasma of animals dying of anthrax infection contained a toxin
which causes symptoms of anthrax when injected into normal guinea pigs. These
observations left little doubt that a diffusible exotoxin plays a major role in the
pathogenesis of anthrax.

One component of the anthrax toxin has a lethal mode of the action that is not entirely
understood at this time. Death is apparently due to oxygen depletion, secondary shock,
increased vascular permeability, respiratory failure and cardiac failure. Death from
anthrax in humans or animals frequently occurs suddenly and unexpectedly. The level of
the lethal toxin in the circulation increases rapidly quite late in the disease, and it closely
parallels the concentration of organisms in the blood.

Production of the anthrax toxin is mediated by a temperature-sensitive plasmid, pX01, of

110 megadaltons. The toxin consists of three distinct antigenic components. Each
component of the toxin is a thermolabile protein with a mw of approximately 80kDa.

Factor I is the edema factor (EF) which is necessary for the edema producing activity of
the toxin. EF is known to be an inherent adenylate cyclase, similar to the Bordetella
pertussis adenylate cyclase toxin.

Factor II is the protective antigen (PA), because it induces protective antitoxic antibodies
in guinea pigs. PA is the binding (B) domain of the anthrax toxin which has two active
(A) domains, EF (above) and LF (below).

Factor III is known as the lethal factor (LF) because it is essential for the lethal effects
of the anthrax toxin. Apart from their antigenicity, each of the three factors exhibits no
significant biological activity in an animal. However, combinations of two or three of the
toxin components yield the following results in experimental animals.

PA+LF combine to produce lethal activity

EF+PA produce edema
EF+LF is inactive
PA+LF+EF produces edema and necrosis and is lethal

These experiments suggest that the anthrax toxin has the familiar A-B enzymatic-binding
structure of bacterial exotoxins with PA acting as the B fragment and either EF or LF
acting as the active A fragment.

EF+PA has been shown to elevate cyclic AMP to extraordinary levels in susceptible cells.
Changes in intracellular cAMP are known to affect changes in membrane permeability
and may account for edema. In macrophages and neutrophils an additional effect is the
depletion of ATP reserves which are needed for the engulfment process. Hence, one effect
of the toxin may be to impair the activity of regional phagocytes during the infectious
process.

The effects of EF and LF on neutrophils have been studied in some detail. Phagocytosis
by opsonized or heat-killed Bacillus anthracis cells is not inhibited by either EF or LF,
but a combination of EF + LF inhibits engulfment of the bacteria and the oxidative burst
in the pmns. The two toxin components also increased levels of cAMP in the neutrophils.
These studies suggest that the two active components of the toxin, EF + LF, together
increase host susceptibility to infection by suppressing neutrophil function and impairing
host resistance.

LF+PA have combined lethal activity as stated above. The lethal factor is a Zn++
dependent protease that induces cytokine production in macrophages and lymphocytes,
and its mechanism of action is slowly becoming understood. The crystal structure of
lethal factor is known to to be a member of the mitogen-activated protein kinase
(MAPKK) family of enzymes that disrupts cellular signaling. Furthermore, the identity of
the human receptor for anthrax PA, named anthrax toxin receptor, has been
demonstrated to be a type I membrane protein that binds directly to PA.

In summary, the virulence of Bacillus anthracis is attributable to three bacterial

components; 1. Capsular material composed of poly-D-glutamate polypeptide; 2. EF
component of exotoxin; 3. LF component of exotoxin. Both the capsule and the anthrax
toxin may play a role in the early stages of infection, through their direct effects on
phagocytes. Virulent anthrax bacilli multiply at the site of the lesion. Phagocytes migrate
to the area but the encapsulated organisms can resist phagocytic engulfment, or if
engulfed, can resist killing and digestion. A short range effect of the toxin is its further
impairment of phagocytic activity and its lethal effect on leukocytes, including
phagocytes, at the site. After the organisms and their toxin enter the circulation, the
systemic pathology, which may be lethal, will result.

Bacillus anthracis coordinates the expression of its virulence factors in response to a

specific environmental signal. Anthrax toxin proteins and the antiphagocytic capsule are
produced in response to growth in increased atmospheric CO2. This CO2 signal is thought
to be of physiological significance for a pathogen which invades mammalian host tissues.

Immunity to Anthrax

Considerable variation in genetic susceptibility to anthrax exists among animal species.

Resistant animals fall into two groups: (1) resistant to establishment of anthrax but
sensitive to the toxin and (2) resistant to the toxin but susceptible to establishment of
disease. This is illustrated in the table below. Neither the source of the inoculum (spores
or vegetative cells or a mixture) nor the route of inoculation (subcutaneous,
gastrointestinal, or inhalational) is stated. The infectious dose of anthrax is expected to
vary widely based on these parameters, as well.

Table 2. The infectious dose of B. anthracis and the lethal dose of toxin varies greatly within animal
species. The data do not specify the route of infection or whether spores or vegetative cells were used
in the inoculum.

Animal Infectious Toxic dose causing Bacteria per ml blood at time

model dose death death
Mouse 5 cells 1000 units/kg 107
Monkey 3000 cells 2500 unit/kg 107
Rat 106 cells 15 units/kg 105

Animals surviving naturally-acquired anthrax are immune to reinfection. Second attacks

are extremely rare. Permanent immunity to anthrax seems to require antibodies to both
the toxin and the capsular polypeptide, but the relative importance of the two kinds of
antibodies appears to vary widely in different animals.

Vaccines composed of killed bacilli and/or capsular antigens produce no significant

immunity. A nonencapsulated toxigenic strain has been used effectively in livestock. The
Sterne Strain of Bacillus anthracis produces sublethal amounts of the toxin that induces
formation of protective antibody.

The anthrax vaccine for humans, which is used in the U.S., is a preparation of the
protective antigen recovered from the culture filtrate of an avirulent, nonencapsulated
strain of Bacillus anthracis that produces PA during active growth. Anthrax immunization
consists of three subcutaneous injections given two weeks apart followed by three
additional subcutaneous injections given at 6, 12, and 18 months. Annual booster
injections of the vaccine are required to maintain a protective level of immunity.

The vaccine is indicated for individuals who come in contact in the workplace with
imported animal hides, furs, bone, meat, wool, animal hair (especially goat hair) and
bristles; and for individuals engaged in diagnostic or investigational activities which may
bring them into contact with anthrax spores. Otherwise, it has been indicated for the
military during the current era of biological warfare.

The vaccine should only be administered to healthy individuals from 18 to 65 years of

age, since investigations to date have been conducted exclusively in that population. It is
not known whether the anthrax vaccine can cause fetal harm, and pregnant women should
not be vaccinated.

A new type of passive vaccine to anthrax is currently on the horizon. This was recently
announced by R.G. Crystal and colleagues from the Medical College of Cornell
University, in the February, 2005 issue of the journal, Molecular Therapy. They
demonstrated that mice vaccinated with a human adenovirus expressing a single-chain
antibody directed against protective antigen (PA) became immune to anthrax within 24
hours of vaccination. This is much quicker than is possible with existing anthrax
vaccines, which are a relatively crude preparation of PA.

Currently available anthrax vaccines have limited use in a bioterrorism attack because
they are active vaccines in which multiple doses are required over several months to elicit
protective immunity against anthrax. Passive vaccines, on the other hand, introduce fully
formed antibodies directly to the body and immunity is achieved much sooner.

In mice receiving the adenovirus-based anti-PA vaccine, PA-specific serum antibodies

were detectable within 24 hours. These antibodies had neutralizing activity that protected
mice from an intravenous lethal toxin challenge administered 1-14 days post vaccination.

Crystal, et al envision a possible scenario wherein both the passive and active vaccine
might be given. Passive vaccines lose their effectiveness fairly rapidly over time, whereas
active vaccines do not. The passive vaccine could provide protection that would last a
couple of weeks, but that would provide a safety margin for development of more active,
long-term immunity stimulated by the active vaccine.

Passive immunotherapy with such adenovirus-based vectors expressing anti-PA antibody,

either alone or in combination with antibiotics, may be a rapid, convenient, and highly
effective strategy to protect against or treat anthrax in a bioterrorism attack.

Also, in cases of anthrax, coadministration of the passive vaccine with antibiotics may
maximize the utility of antibiotic therapy. Coadministration would counter the effects of
lethal toxin, and likely prolong the time frame for effective antibiotic treatment and/or
reduce the amount of antibiotic therapy required.

Treatment of Anthrax

Antibiotics should be given to unvaccinated individuals exposed to inhalation anthrax.

Penicillin, tetracyclines and fluoroquinolones are effective if administered before the
onset of lymphatic spread or septicemia, estimated to be about 24 hours. Antibiotic
treatment is also known to lessen the severity of disease in individuals who acquire
anthrax through the skin. Inhalation anthrax was formerly thought to be nearly 100% fatal
despite antibiotic treatment, particularly if treatment is started after symptoms appear. A
recent Army study resulted in successful treatment of monkeys with antibiotic therapy
after being exposed to anthrax spores. The antibiotic therapy was begun one day after
exposure.

Anthrax and Biological Warfare

The inhalation of anthrax spores can lead to infection and disease. The possibility of
creating aerosols containing anthrax spores has made B. anthracis a chosen weapon of
bioterrorism. Several powers may have the ability to load spores of B. anthracis into
weapons. Domestic terrorists may develop means to distribute spores via mass attacks or
small-scale attacks at a local level.

As an agent of biological warfare it is expected that a cloud of anthrax spores would be

released at a strategic location to be inhaled by the individuals under attack. Spores of B.
anthracis can be produced and stored in a dry form and remain viable for decades in
storage or after release.

There is no evidence of person-to-person transmission of anthrax. Quarantine of affected

individuals is not recommended. Anthrax spores may survive in the soil, water and on
surfaces for many years. Spores can only be destroyed by steam sterilization or burning.
Chemical disinfection of buildings is problematic. The U.S. Navy Manual on Operational
Medicine and Fleet Support, entitled Biological Warfare Defense Information Sheet states
"Disinfection of contaminated articles may be accomplished using a 0.05% hypochlorite
solution (1 tbs.. bleach per gallon of water). Spore destruction requires steam
sterilization."

Anthrax spores are killed by boiling (100oC or 212oF) for 30 minutes (the actual reported
time is considerably less). If boiling as a means of disinfection, the spores must be in
liquid suspension to ensure killing, and in a sealed container to avoid aerosolization or
vaporization of droplet nuclei containing spores.

An infection of local animal populations such as sheep and cattle could follow a
biological attack with spores. Infected animals could then transmit the disease to humans
through the cutaneous, intestinal or inhalation route by spores from a contaminated
animal, carcass or hide.

A segment of the U.S. military population has been vaccinated against anthrax. Anthrax
vaccine consists of a series of six doses with yearly boosters. The first vaccine of the
series must be given at least four weeks before exposure to the disease. This vaccine
protects against anthrax that is acquired through the skin and it is believed that it would
also be effective against inhaled spores in a biowarfare situation. Of course, a vaccinated
military population would be needed to respond to a bioterrorist attack with anthrax
spores.