Overview of Bacillus anthracis
Overview of Bacillus anthracis

Bacteriology

Overview of Bacillus anthracis

Bacillus anthracis the most well-known pathogen in the Genus Bacillus is the cause of a severe zoonotic illness known as anthrax. Anthrax...

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This article writter by MN Editors on January 24, 2022

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Bacillus anthracis the most well-known pathogen in the Genus Bacillus is the cause of a severe zoonotic illness known as anthrax. Anthrax is a primary disease that affects domestic and wild herbivores. It is among the most frequently encountered bioterrorism agents that has been implicated before in the Sverdlovsk the anthrax outbreak of 1979 as well as US postal system attacks in 2001.

What is Bacillus anthracis?

Bacillus anthracis is only the pathogenic species that is obligately Bacillus and the agent responsible of anthrax. It is a widespread disease that affects livestock , and sometimes in humans.

Anthrax is referred to as an zoonotic disorder because the disease is transmitted between humans and animals through different methods. It is a Gram positive spore-forming bacterium which is typically found in soil. However, like the other Bacillus species, it can result in various forms of infection if it enters the gastrointestinal, respiratory, or cutaneous areas of humans.

The main mode of transmission for B. anthracis happens via the spores. The spores are transformed into vegetative cells when they enter within the human body host , and result in infections. They are extremely resistant to the adverse environmental conditions However, the cells that are vegetative from B. anthracis are unable to survive in basic environments such as bulk soil and water.

B. anthracis is part the B. cereus group of Bacillus species, which includes other pathogens with opportunistic characteristics, like B. cereus as well as B. Thuringiensis. The possibility to use B. anthracis to be bioweapon or bioterrorism threat has been debated and even feared for quite a long period of time following the use this bacteria to kill livestock World War I. It is now known that people are much more resistant B. anthracis than other herbivores and the levels of infection by the bacterium found in humans are also extremely high.

Yet, anthrax-related cases in humans have been reported in the general population because of close proximity to affected animals by handling affected domestic animals. B. anthracis first identified from domestic animals infected through Cohn around 1872.

The term ‘anthracis’ for the species is an abbreviation for the illness, anthrax which is in turn derived from the Greek word meaning coal, which is derived from the formation of black coal-like skin Eschars. B. anthracis is extensively studied because of the pathogenicity the species as well as its potential as bioterrorism agent.

History of Bacillus anthracis

French doctor Casimir Davaine (1812-1882) demonstrated anthrax-related symptoms almost always caused by bacteria called B. anthracis. German physician Aloys Polender (1799-1879) is the one who was credited with discovering. B. anthracis is the first bacteria that was conclusively proven to cause illness, in the hands of Robert Koch in 1876. The term “anthracis” comes an abbreviation of the Greek anthrax (anthrax) which means “coal” and referring to the most commonly used type of illness, cutaneous anthrax which is where black, large skin lesions form. In during the nineteenth century Anthrax was a disease which brought about a number of important medical advancements. The first vaccine made with living microorganisms came from Louis Pasteur’s vet anthrax vaccine.

Classification of Bacillus anthracis

The Genus Bacillus is part of the Bacillaceae family. Bacillaceae which includes other genera that are classified by their molecular and phenotypic characteristics. More than 142 species that belong to the Bacillus genus Bacillus that are further divided into manageable groups based on the foundation of the 16S sequences of rRNA.

B. anthracis is part of B. anthracis is part of the B. cereus family of Bacillus species as well as other pathogens, or pathogens that are opportunistic, such as B. cereus as well as B. thuringiensis. The genomes of the B. cereus group are highly conserved and have dimensions of 5.2-5.5 Mb. They also share identical 16S rRNA gene sequences. The distinction and classification from B. anthracis in comparison to other species of the group could be established through an amplified polymorphism in fragment length analyses since they have the same 16S sequences of rRNA genes.

There are 89 distinct species that belong to B. anthracis, which have been isolated from various regions around the globe and are utilized for various objectives. B. anthracis has a monomorphic form, and has limited genetic diversity and no evidence of DNA transfer lateral. The strains in this species are phenotypically and genetically heterogeneous generally, but certain strains are closer to each other, since they are interspersed phylogenetically on the chromosomes level. There are some differences that could be noticed as the life cycle of bacteria is centered around animals as hosts, and its environment.

Here is the taxonomical classification for B. Anthracis

DomainBacteria
PhylumFirmicutes
ClassBacilli
OrderBacillales
FamilyBacillaceae
GenusBacillus
SpeciesB. anthracis

Genome structure

B. anthracis is only one chromosome, which is a circular 5,227.293-bp DNA molecule. Also, it has two extrachromosomal, circular, double-stranded DNA plasmids: the pXO1 and the pXO2. Both pXO1 as well as the pXO2 plasmids are essential for full virulence. They represent two distinct families of plasmids.

FeatureChromosomepXO1pXO2
Size (bp)5,227,293181,67794,829
Number of genes5,508217113
Replicon coding (%)84.377.176.2
Average gene length (nt)800645639
G+C content (%)35.432.533.0
rRNA operons1100
tRNAs9500
sRNAs320
Phage genes6200
Transposon genes18156
Disrupted reading frame3757
Genes with assigned function2,7626538
Conserved hypothetical genes1,2122219
Genes of unknown function65785
Hypothetical genes87712251

pXO1 plasmid

The pXO1 genome (182 kb) includes the genes that encode the components of anthrax’s toxin including pa (protective antigen or PA) and lef (lethal factor lethal factor, LF) as well as the cya (edema factor, the EF). These components are located in the 44.8-kb area of pathogenicity (PAI). The lethal toxin is combination of PA and LF and the Edema Toxin is a mix of PA and EF. The PAI also has genes that encode the transcriptional activator AtxA and a Repressor PagR both of which control the expression of anthrax toxin gene.

pXO2 plasmid

The pXO2 gene encodes a five-gene operon (capBCADE) that synthesizes a poly-g D-glutamic acid (polyglutamate) capsule. The capsule permits B. anthracis avoid the immune system of the host by shielding itself from the phagocytosis process. The capsule operon is stimulated by transcriptional regulatory regulators AcpA and AcpB which are located in the pathogenicity island of pXO2 (35 km2). AcpA as well as AcpB transcription is controlled by AtxA from pXO1.

Types of Anthrax

The type of illness that a person suffers from is dependent on the way in which anthrax gets into their body. The majority of the time, anthrax enters the body via either the skin or lungs or the digestive system. The various forms of anthrax could be spread to other parts of the body and lead to death if not treated by antibiotics.

1. Cutaneous anthrax

Cutaneous anthrax is considered to be the most frequent type of anthrax It is thought by many to be least hazardous. The infection usually begins one to seven days following exposure.

When anthrax spores infiltrate the skin, often by way of a cut or scrape the skin, an individual can develop anthrax that is cutaneous. It can occur when an individual handles infected animals or other animal products that have been contaminated such as hides, wool or hair. Cutaneous anthrax can be found in the neck, head hands, forearms, and the neck. It can affect the skin and tissues surrounding the site of infection.

If treatment is not provided, up to 20% of those suffering from anthrax in the cutaneous area suffer fatal deaths. However, with appropriate treatment, the majority of patients suffering from cutaneous anthrax live.

2. Inhalation anthrax

Inhalation anthrax is believed to be the deadliest type of anthrax. The infection usually manifests within the first week following exposure, however it could last for up to two months.

If a person inhale anthrax spores they may be diagnosed with anthrax inhalation. Personnel who work in workplaces like wool mills, slaughterhouses and tanneries can breathe in spores working with animals infected, or products of animals that are contaminated from infected animals. The first signs of anthrax inhalation are within the lymph nodes of the chest, before spreading to the the body, eventually causing extreme breathing issues and shock.

If not treated, inhalation anthrax can be fatal. But even with aggressive treatment approximately 50% of sufferers can survive.

3. Gastrointestinal anthrax

Gastrointestinal anthrax has not been documented within the United States. The infection usually begins one to seven days following exposure.

If a person consumes raw or undercooked food items from an animal that is infected by anthrax may develop digestive anthrax. When ingested, the spores from anthrax may affect the upper digestive tract (throat and the esophagus) as well as stomach and the intestines, causing a array of symptoms.

Without treatment, over 50% of patients suffering from intestinal anthrax will die. But, with the right treatment about 60% are able to live.

4. Injection anthrax

This kind of infection is not documented within the United States.

Recently, a different kind of anthrax has been discovered among heroin users who inject drugs in northern Europe.

The symptoms may be similar to symptoms of the cutaneous anthrax. However, there is a possibility of inflammation deep within the skin or inside the muscle in which the drug was administered. Anthrax from injections can spread through the body quicker and become more difficult to detect and manage. Many other common bacteria can cause infections of the injection site and skin So the presence of a skin or injection site infection for a user of a medication doesn’t necessarily mean that the patient has anthrax.

Habitat of Bacillus anthracis

  • Bacillus anthracis can be described as an aerobic bacterium that produces endospores which is found in a vast variety of natural ecosystems.
  • It is the only obligatory pathogen that can be found in animals, such as mammals, humans as well as insects. The primary habitat, however, is the soil and it can transfer to other ecosystems in forms of spores.
  • While the bacteria may were isolated from various habitats, all the environments they have been isolated from are not their habitats.
  • B. anthracis just like other Bacillus species thrives in soils that are acidic as well as alkaline environments within a large temperature range.
  • B. anthracis, found in the natural environment outside the body of the host, is present as spores, as the environmental conditions becomes more difficult.
  • The life cycle and habitat that are exhibited by B. anthracis is described by the micro-cycling of the soil-based bacteria and the frequent multiplication phase for animals.
  • Although there isn’t enough evidence to support what causes the proliferation of B. anthracis within soil It is believed that the spores from B. anthracis could develop into a vegetative cell and multiply if conditions in the environment are favorable.
  • The capacity to allow B. anthracis live in these diverse environments is due to the presence of endospores that are highly resistant. They are more resistant to adverse conditions, certain chemicals and antimicrobial agents than vegetative cells.
  • The spores tend to be small and easily dispersed through dust or in the creation of aerosols.
  • Thus, the spores are absorbed into in the organism of their host (mostly herbivores) in which they germinate to create vegetative cells. The vegetative cells may then be able to enter new niches, similar to the human body, but differ from animals.
  • Apart from that, there are additional ways to produce toxin, which allows bacteria to compete with other bacteria, and to take over new niches or ecology.
  • The prevalence in B. anthracis can be found more frequent in warmer climates. This is believed that it is due to the connection between temperature and activity in the water and the rate of the sporulation of the bacteria that are shed by the body of the affected animal.
  • The temperature as well as the water activity can also affect the germination process of spores but to a lesser extent.
  • One of the main reasons to the existence in B. anthracis across a range of conditions is the capacity for the spores to live for a long time even without reservoirs from animals.

Morphology of Bacillus anthracis

  • These cells from B. anthracis comprise Gram positive rods, which are aerobic, facultative anaerobes, which are capable of forming spores and capsulation.
  • The cells of sizes that range between 1.0-1.2 inches in size and 3.0-5.0 millimeters in length appear either as a pair or singly. In clinical samples however, cells could form shorter chains.
  • All cells that belong to B. anthracis are dotted with cylindrical or ellipsoidal spores which are found paracentrally or subterminally within the vegetative cells.
  • The spores are not responsible on the expansion of sporangia because they are usually on the oblique side of it.
  • Phenotypically speaking, B. anthracis appears like other Bacillus species such as B. cereus as well as B. thuringiensis but, unlike these species, B. anthracis does have no flagella, and therefore isn’t a movable species.
  • The outer cover the outer covering of B. anthracis can be distinguished by a capsule, an extensive the peptidoglycan layer and lipoteichoic acids and crystal cells’ surface proteins (S Layer).
  • The capsule of B. anthracis consists of poly-g-D-glutamic acids which is encoded through three different plasmid gene.
  • A capsule in B. anthracis forms among the virulence factors , since the strains without the capsule are not virulent.
  • The capsule is safe and non-immunogenic since it does not stimulate the immune system of the host.
  • Underneath the capsule lies the S-layer or the surface layer made up of proteins. they’re not glycosylated.
  • The cell wall polysaccharides that are present on the cell’s wall serve in anchoring the cell’s surface on the wall of cells. The cell wall is comprised of polysaccharides such as galactose, N-acetylglucosamine, as well as N-acety.
  • The connections within the cell wall are made up of mesodiaminopimelic acid that connects the diamino acids of one subunit to the D’alanine subunit.
  • The genomic sequence for B. anthracis is tripartite and comprises one circular chromosome as well as two circular virulent plasmids within the cytoplasm. The genome is 5227293 Bp long and contains 508 sequences that code for proteins.
  • The genome nucleotide is made up of around 60 percent adenine, thymine and adenine units, with only 40% of the guanine as well as the cytosine.
  • The chromosome’s composition leads to a greater buoyant density and lower melting point of DNA units.
  • The plasmids are called pXO1 and the pXO2 which code for many different genes , including capsule and toxin production genes.

Cultural Characteristics of Bacillus anthracis

  • Artificial growth as well as cultivation of B. anthracis is typically accomplished by the isolation by the bacteria of dead carcasses or animal products. They can also be isolated from soil samples, where they are present as spores.
  • The identification from B. anthracis is done on blood, nutrient or selective agars, based on the source of the sample.
  • There isn’t a reliable enrichment technique for the isolation from B. anthracis. However, significant selective isolation can be achieved using polymyxin-lysozyme-EDTA-thall Acetate (PLET) Agar.
  • As with most Bacillus species The nutrient requirements of B. anthracis simple, so the growth of B. anthracis can be accomplished in simple environments using glucose as the main source of carbon, and ammonium salt as nitrogen source.
  • A casein-based hydrolyzed medium containing glucose and thiamine, tryptophan and various salts is commonly used to study B. anthracis’s physiology and gene expression.
  • The morphology of the colony as well as its development, is affected by many factors such as the germination of spores the composition of the medium, and the conditions of incubation.
  • A heat treatment of spores prior to their growth encourages the sprouting of spores as well as the growth of vegetative cells.
  • B. anthracis has the capacity to be a facultative anaerobe, therefore, the highest development is seen when it is incubated for a few hours under CO2 of 5-7%.
  • The shape and morphology of the colonies as well as the surface of colonies are also influenced by the existence or lack of capsules, as mucoid colonies are formed when colonies are capsulated.
  • The development of B. anthracis is possible between 5degC and 45degC, with the highest growth occurring at 37degC dependent on the source of the bacteria.
  • Colonies of B. anthracis appear like the different species of Bacillus and are identified by spikes or tails according to the inoculation streaks. They can also be extremely tenacious.
  • The spread of B. anthracis could be seen as a swarming phenomenon across the media , instead of as isolated colonies. This can be prevented by raising the agar content of the medium.
  • In the case of liquid media B. anthracis usually develops as planktonic cells however, pellicles may develop during static incubation and adhesion to solid surfaces could be observed.
  • The development of B. anthracis in artificial media occurs in two phases , starting with vegetative growth, which leads to the formation of spores when the culture ages.

Here are some of the cultural traits from B. anthracis in various media of culture:

1. Bacillus anthracis on Nutrient Agar (NA)

Based on the type of sample, B. anthracis may be cultivated on a variety of growth mediums, ranging that range from a simple medium such as Nutrient agar, to a more complicated and selective medium such as PLET agar. The colonies that grow on B. anthracis are comparable to those of different members in the B. cereus group , and may require a different method to determine the species of the organism. On NA, the colonies that belong to B. anthracis are irregularly shaped, with margins of undulating and fimbriate edges or crenates.

If the conditions for growth do not favor capsule development, colonies show irregular edges and a rounded “ground-glass” look. The colonies range from creamy to white with huge dimensions (2-7 millimeters in size) However, the size of colonies that are young may be smaller. The colonies that belong to B. anthracis differ than other Bacillus species because they develop clusters or tails along those lines that are inoculated. Some colonies could create standing peaks, when pulled using loops.

The colony’s surface is usually matte or granular texture, however dry and smooth colonies may be present. Colonies in solid media favor capsule synthesis that results in mucoid colonies that have a huge capsule that results in colonies that are thick (up to 3um in size).

2. Bacillus anthracis on Blood Agar

The blood agar that is used in to isolate B. anthracis was made using 5% sheep’s blood to the nutrient the agar. These colonies from B. anthracis are not hemolytic or g-hemolytic. This aids in the distinction from B. anthracis in comparison to other Bacillus species, such as B. cereus or B. Thuringiensis. The colonies are flat or slightly convex with irregular edges and a appearance of glass are seen. They often feature comma-shaped projections that extend from the edges of the colony, resulting as medusa heads. The size of colonies is significantly smaller than those on Nutrient Agar. The range of size is between 2 and 4 millimeters. However, the size could increase on an additional day.

Bacillus anthracis on Blood Agar
Figure: Bacillus anthracis, which were cultured on sheep blood agar (SBA) medium, for a 24-hour time period, at a temperature of 37°C. Image Credit: Todd Parker

3. Bacillus anthracis on PLET Agar

PLET agar is a highly one-stop solution for the separation of B. anthracis in environmental samples including animal products, and clinical specimens. The greater concentration of EDTA on this media hinders the expansion of Staphylococcus aureus, as well as B. cereus. B. cereus’ colonies B. anthracis appear to be roughly circular, creamy white with a glass-like texture. In the event that the B. cereus colonies are visible, B. cereus have been observed in the same way, they are usually smaller than those of B. anthracis. The capsule could be visible on the surface within 48 hours after incubation.

Bacillus anthracis on PLET Agar
Figure: Bacillus anthracis cultured on mannitol, egg yolk, polymyxin agar (MEP) medium, for a 24-hour time period, at a temperature of 37°C. Image Credit: Todd Parker

Biochemical Characteristics of Bacillus anthracis

The biochemical properties that characterize B. anthracis may be summarized according to:

Biochemical Characteristics B. anthracis
Capsule Capsulated with a poly-γ-glutamic acid capsule.
Shape Rod 
Gram Staining Gram-Positive
CatalasePositive (+) 
Oxidase Negative (-) 
Citrate Positive (+)
Methyl Red (MR)Negative (-)
Voges Proskauer (VR)Positive (+)
OF (Oxidative-Fermentative)Facultative Heterofermentative
CoagulasePositive (+)
DNaseNegative (-)
UreaseNegative (-)
GasNegative (-)
H2SNegative (-)
HemolysisNon-hemolytic
Motility Non-motile as they lack flagella.
Nitrate Reduction Positive (+)
Gelatin HydrolysisPositive (+)
Pigment Production Negative (-)
Indole Positive (+)
TSIA (Triple Sugar Iron Agar)Alkali/Alkali (Red/ Red)
SporeEndospore-forming
Penicillin SusceptibilitySusceptible

Fermentation

Substrate B. anthracis
AdonitolNegative (-)
Arabinose Negative (-)
Cellobiose Negative (-)
DulcitolNegative (-)
Fructose Positive (+)
Galactose Negative (-)
Glucose Positive (+) Facultative heterofermentative
Glycerol Negative (-)
GlycogenPositive (+)
HippurateNegative (-)
Inulin Negative (-)
Inositol Negative (-)
Lactose Negative (-)
MalonatePositive (+)
Maltose Positive (+)
Mannitol Negative (-)
Mannose Positive (+)
MelibioseNegative (-)
Pyruvate Negative (-)
Raffinose Negative (-)
Rhamnose Negative (-)
Ribose Positive (+)
Salicin Negative (-)
Sorbitol Negative (-)
Starch Positive (+)
Sucrose Positive (+)
Trehalose Positive (+)
Xylose Negative (-)

Enzymatic Reactions

EnzymesB. anthracis
Acetoin Positive (+)
Acetate UtilizationPositive (+)
Tyrosine Hydrolysis Negative (-)
LecithinaseNegative (-)
Casein HydrolysisPositive (+)
Esculin HydrolysisPositive (+)
Lysine decarboxylasePositive (+)
Ornithine DecarboxylaseNegative (-)
Phenylalanine DeaminaseNegative (-)

Virulence Factors of Bacillus anthracis

Bacillus anthracis is a obligate pathogenic bacterium which is the cause of the disease anthrax. It’s found primarily in herbivores, but less frequently in humans. However, animals such as dogs, pigs, cats as well as chickens are not susceptible to anthrax however, the bacteria can spread to birds once eating scavenging birds such as vultures on animals who have died from an infection. The primary element that allows the bacteria to live and cause infection is the capability for the bacteria to develop spores. The spores are immune to diverse environmental threats.

In addition to the three main factors that influence the virulence associated with B. anthracis There are other secreted or not secreted factors that influence the host-pathogen interaction. Certain proteases inhibit the immune system by breaking down the antimicrobial proteins. Other proteases such as InhA1 may degrade host tissue and cause increased barrier permeability. B. anthracis is a rare virulence, manifesting in three primary factors that contribute to virulence:

1. Capsule

B. anthracis creates the poly-glutamic acid (PGA) capsule that offers the bacterium with protection from the phagocytosis that is common to many pathogenic bacteria. Its negative charges the membrane hinders defense of the host by preventing phagocytosis of plant cells of macrophages as well as various immune cells. The capsule is formed through spores germination under the influence of serum as well as increased CO2 by openings in the surface of the spores as a pores, which can coalesce prior to the exosporium is sloughed off and the outgrowth of the encapsulated cells.

Because the capsule is on the outside of the S layer and does not require the S-layer to be attached to the cell’s surface. The capsule’s synthesis is assisted through three membrane-associated enzymes that are encoded within the 60-MDa pXO2 the plasmid. The genes involved in enzyme’s synthesis are capB, capC and capA, which encode the 44, 16 and 46kDa proteinsrespectively.

Capsules of B. anthracis remarkably immunogenic and antiphagocytic , which shields the bacilli in the absence of immune surveillance. Additionally, the capsule activates caspase-1 , which triggers an expulsion of interleukin-1b in differentiated T-cells as well as human monocyte-derived dendritic cells. The function that capsule protein enhances is aided by antigens, proteins and toxic plasmid-coded toxicants.

2. Endotoxin

Bacillus anthracis is responsible for two distinct endotoxins, which are released as three components: the protective antigen (PA) and edema factor (EF) the lethal factors (LF). The 3 proteins encoded in the virulence plasmid , pXO1, which triggers hemorrhage and necrosis, and edema. The protective antigen acts as the cellular binding component of the toxins, while the lethal factor as well as the edema factors are the catalytic elements.

The Edema factor can be described as a mature protein, with 767 residues and an estimated molecular mass of 89 kDa. It is a calmodulin-dependent adenylate cycler that converts intracellular ATP into the cAMP. The amino terminal component of the factor is characterized as a stable polypeptide which can compete with LF in binding to the antigen that protects. Lethal factor is mature that has 776 residencies and a molecular weight of 85kDa it is an zinc metalloprotease which is able to cleave and inhibit the protein kinase that is activated by nitrogen. Similar to the edema factor, a lethal one also contains an aminoterminal component which allows the binding of the protein to PA.

The PA that is released is proteolytically cleaved or furin-like proteases, resulting in two fragments: PA63 as well as PA20. Elimination of PA20 eliminates the steric hindrance. This allows PA63 to be a LF/EF binding component. The cleavage produces residues on PA63 which are able to be able to bind three or four EF or LF molecules, while retaining the steric hindrance between toxin molecules. The binding ultimately results in the formation of lethal toxic (LT) and the edema toxin (ET). These toxins play an essential part in the response to a variety of stimuli such as mitogens, proinflammatory cytokines, as well as heat shock.

Pathogenesis of Bacillus anthracis

The process of infection for B. anthracis starts by consuming spores. In animal cases is transmitted in the soil, whereas in humans, it is passed by animals following repeated exposure. The unusual virulence of this B. anthracis bacteria can be attributed to factors that facilitate the persistence of the bacteria and its capability to cause the destruction of host cells throughout its life cycle of infection. The pathogenesis that is the main cause that causes B. anthracis is described as follows;

1. Entry 

The main infectious form of B. anthracis is its spore which enters inside the patient from the environment via various ways. The spores are invulnerable to diverse environmental conditions and may develop into vegetative forms when the conditions are favorable.

Macrophages are able to rapidly phagocytose bacteria inside the body of the host Some spores are killed by macrophages. Other spores, particularly those that are introduced into the body through inhalation, can survive the phagocytosis process and are carried to the mediastinal lymph nodes through the lymphatic system.

The phagocytosed spores need a few days of incubation before they can germinate. The latency is seen in the respiratory version of the disease, but however not with the more cutaneous type. The germination process is initiated by increased CO2 levels and also the temperatures of the patient.

2. Invasion

The germination of spores into cells that can grow followed by activation of the capsule and toxin gene expression in the plasmids of this organism. The capsule plays a role in the phagocytosis resistance as due to the negative charge found on it. The toxins are released as three protein components which undergo cleavage before binding to form the toxins in the end.

Protective antigen (PA) attaches to molecules of a specific membrane protein in the host cell, which is in this case Anthrax Toxin Receptor (ATR). PA is then broken down by proteases to form two parts that one of which is bound with one toxin components or both. The resulting complex is released through the cell via endocytosis that is mediated by receptors, and eventually to an acidified endosome following the change in conformation of the toxin molecules.

The edema toxin binds to the host protein calmodulin , and develops into an active adenylylcyclase. The enzyme increases levels of cAMP, which causes hypovolemic shock. The edema toxin can increase the vulnerability of the host to infection through activating chemotaxis in neutrophils in humans. The lethal toxin subsequently cleaves the the mitogen-activated protein-kinase family blocking certain signaling pathways, and thus increasing the levels of cytokines responsible for triggering shocks like TNFa and the IL-1b.

In the initial stages of infection, as a result of the synergistic impact from both toxins, decrease in the production of pro-inflammatory cytokines is observed, which facilitates the growth of bacterial colonies within the host. Edema and lethal toxins can cause an inflammatory vascular reaction, though the severity of the shock could differ. Lethal toxin triggers a non-hemorrhagic, cytokine-independent, vascular hypoxic necrosis. Edema toxin causes a generalized cAMP-mediated blood vessel dysfunction.

While the primary target of ET are hepatocytes, epithelial cells also suffer from attack during the cutaneous manifestations of the disease. This results in the phenotypes that characterize the lesions. The infection is ongoing the bacteria move into blood with final levels of the bacteria reaching 107 to the level of 109 cells/ml for susceptible hosts.

Pathogenesis of Bacillus anthracis
Figure: Pathophysiology of Anthrax. Image Source: NEJM.

Clinical Manifestation of Bacillus anthracis

The disease that is caused due to B. anthracis can be known as anthrax, which manifests in three distinct forms based on the entry route for the bacteria. The most prevalent type of anthrax is cutaneous anthrax, which represents 90 percent of all cases of human. Two other types of anthrax include gastrointestinal anthrax and pulmonary anthrax, also known as inhalation anthrax. B. anthrax can also be connected to meningitis.

1. Cutaneous Anthrax

The time of incubation for cutaneous anthrax can be as short as 2 days, but in certain cases could extend to two weeks. The majority of exposure in the cutaneous area is due to occupational or through handling animals infected or laboratory material. The spores infiltrate the body through a break within the skin. Eventually, within 2 to 5 weeks, skin is covered with the initial lesions.

The first lesions are identified by a psoriasis-free papules, which transform into an ulcer over the course of 24 to 36 hours, with accompanying vesicles. The process of ulceration occurs followed by the drying out to form a traditional black eschars, with eventual an expansion of the vesicle. The lesions may contain pus, in cases of secondary infections caused by bacteria that are pyogenic such as Staphylococcus aureus.

B. anthracis cells remain confined to the lesion when it is the case of anthrax uncomplicated, but lymph nodes could also develop. The eschar begins to slowly disappear within one to six weeks after the first appearance of the lesion, regardless of treatment. If untreated anthrax, 20percent or less of patients may develop septicemia, and eventually die. When using the appropriate antibiotics however the mortality rate is not more than 1percent.

2. Gastrointestinal Anthrax

Gastrointestinal Anthrax occurs as a result of the infiltration of spores of bacteria through the ingestion of raw meat of animals that are infected with B. anthracis. The duration of incubation is comparable to that of a skin infection and the same characteristic eschar develops in the wall of the caecum or terminal ileum. Gastrointestinal anthrax may manifest in two types of symptoms: abdominal and oro-oesophageal.

When abdominal anthrax, symptoms such as nausea vomiting, anorexia and fever can be observed. As the disease worsens, patients experience severe abdominal pain and haematemesis, bloody diarrhea can result in septicemia and eventually death. If you suffer from oro-oesophageal Anthrax, symptoms may include dysphagia, sore throat and fever as well as swelling. Patients can develop massive ascites in two to four days following the beginning discomfort in the abdomen.

3. Pulmonary Anthrax

Pulmonary anthrax comprises about 2-5 percent of all instances of anthrax. The pulmonary anthrax resulted from the inhalation and inhalation of aerosolized spores. The illness starts with flu-like symptoms, moderate fever, fatigue and malaise that last for about a week after an initial inhalation.

The prodromal phase begins up to 48 hours, and is followed by the development of an acute infection that is characterized by an increase in temperature and cyanosis. In the pulmonary system, it is regarded as to be a point of entry instead of a site of pathology primary; therefore it is absorbed by alveolar macrophages , and later moved to mediastinal lymph nodes instead of creating pneumonia. Within 1-3 days after initial symptoms, the disease can progress into systemic dispersal that is accompanied by diaphoresis, fever chills and shock.

4. Meningitis

Meningitis is a symptom of anthrax that occurs in the final stage of the other types of anthrax. The symptoms manifest quickly and are accompanied by consciousness, however the progression is very slow. The symptoms of clinical illness include presence of blood in cerebrospinal fluid, and eventually the loss of shock or even death.

Lab Diagnosis of Bacillus anthracis

The diagnosis for clinical B. anthracis is confirmed through the visualisation and the culture of B. anthracis in the clinical samples. A diagnosis for anthrax may be established using conventional methods or molecular techniques. The specimens used in the determination for B. anthracis comprise the swabs used to collect fluid from the vesicular system in the case of cutaneous anthrax.

1. Cultural and Biochemical Identification

This is a common method to identify B. anthracis based on expansion on specific media, hemolysis tests, staining of the capsule, motility tests as well as vulnerability to penicillin. To determine the precise isolation from B. anthracis using PLET agar for the detection for B. anthrax based on the cultural traits.

A capsulated cell’s presence may be utilized to determine the identity of B. anthracis using M’Fadyean staining using polychrome Methylene Blue. The standard method of diagnosing of anthrax faces some difficulties because of the phenotypic and genetic similarities with different Bacillus species. The test for hemolysis can be used to distinguish B. anthracis and B. cereus which is b-hemolytic.

2. Antigen-based Methods

The detection of antigens by immunoassay is a second method of the identification of B. anthracis. The most commonly used antigens for these tests are the glycoprotein BclA found in the exosporium, extracellular antibody EA1 of the S layer as well as the protective antigen of the anthrax toxin along with the poly-Dglutamine capsule.

The choice of the target antigen depends on the kind of specimen being examined as various antigens can be found in vegetative cells as well as the spores. The most commonly used immunoassays to determine B. anthracis diagnosis include flow cytometry tests and luminescent adenylate-cyclase tests.

3. Molecular methods

Methods that are based on molecular analysis like PCR that utilize the DNA amplification process allow for the identification of B. anthracis, without cultivation of the bacteria, making them more secure than traditional methods. The most commonly used genetic markers for detection of B. anthracis are found on the virulence-related plasmids PXO1 as well as the pXO2.

The genes that are used include the codon component of anthrax toxin as well as the capsule. The analysis of these genes provides information about the virulence and toxicity that the bacteria. There are however difficulties with these genes since the plasmids may be lost or transferred to different Bacillus species.

Treatment of Bacillus anthracis infections

  • The treatment for anthrax isn’t difficult since the bacteria is insensitive to a variety of antibiotics, including penicillin, erythromycin, ciprofloxacin vancomycin and penicillin. It is intolerant to cephalosporins and sulfonamides and trimethoprim.
  • Penicillin is the medication of choice since resistance against penicillin hasn’t been observed within naturally-occurring strains.
  • However, the fast-paced course of the disease, even when treated with antibiotics has led to high death rates for pulmonary anthrax.
  • Antitoxin therapy has been researched and utilized to stop the rapid development of the disease as well as elimination of toxic substances.
  • Presently treatments using ciprofloxacin amoxicillin and doxycycline are advised in cases that are mildly affected by anthrax on the skin.

Prevention of Bacillus anthracis infections

  • Human-to-human contagion cases have been not documented in the case of anthrax, which suggests that the main type in the infection process involves the spore.
  • This means that the disease can be prevented by maintaining good hygiene and security during handling of animals that are infected.
  • In addition, active vaccination is crucial for anthrax pre-exposure prevention. The only toxin-based vaccine against B. anthrax that has been approved by FDA is BioThrax.
  • While it is designed to be used prior to exposure however, it can be beneficial for post-exposure prophylaxis as well.
  • Materials and instruments that have been contaminated for patients suffering from anthrax must be disposed of in a manner that is autoclaved or burned according to the standard procedure.

B. anthracis as a bioterrorism agent

  • The spores that are resistant to B. anthracis that have the potential of forming aerosols have the potential to be used as a bio-terror weapon in the war.
  • Inhaling spores can be risky as the first signs of the disease are similar to those of flu, making initial diagnosis difficult.
  • The worry about B. anthracis being bioweapons has grown since it was originally created for use in World war I and II. in 2001, envelopes with B. anthracis was delivered by mail to various government officials in the United States which are also classified as bioterrorism.

Laboratory research on Bacillus anthracis

Tea’s constituents like polyphenols are able to block the activity and B. anthracis as well as its toxin in a significant way. Spores however, aren’t affected. In addition, adding milk into the tea totally blocks its antibacterial action against anthrax. The activity against B. anthracis strain in the lab does not show that drinking tea has an effect on the process of infection because it is unclear what happens to these polyphenols and distributed throughout the body. B. anthracis is cultivated on PLET agar which is a differential and selective media that is specifically designed to target B. anthracis.

Recent research

The advancements in methods for genotyping have resulted in enhanced genetic analysis to detect variations and relatedness. Methods used include multiple-locus variable-number tandem repeat analysis (MLVA) and typing systems that employ canonical single-nucleotide polymorphisms. The Ames ancestor genome was sequenced in 2003[9] which contributes in the identification of genes responsible for the pathogenesis of B. anthracis. Recently, B. anthracis isolate H9401 was identified from the body of a Korean sufferer suffering from gastro anthrax. The aim for Korea Republic of Korea is to utilize the strain to create a test strain to create an anti-anthrax recombinant vaccine.

The H9401 strain that was isolated from Korea Republic of Korea was sequenced using GS-FLX 454 technology and analysed using a variety of bioinformatics tools that align, annotate and evaluate H9401 against the other B. Anthracis strains. The level of coverage in the sequencing indicates an atomic ratio of pXO1:pXO2:chromosome as 3:2:1 that is similar to Ames Florida and Ames Ancestor strains. H9401 shares 99.679 percent sequence homology to Ames Ancestor with an amino acid sequence homology of 99.870 percent.

H9401 has a circular-shaped chromosome (5,218,947 bp , with five 480 expected ORFs) as well as the pXO1-related plasmid (181,700 bp with 202 predicated ORFs) and the pXO2 plasma (94,824 Bp with 110 expected ORFs). When compared against H9401, which is the Ames Ancestor chromosome above, the H9401 chromosome is 8.5 km smaller. Due to its significant pathogenecity and the sequence similarity in comparison to Ames Ancestor, H9401 will be utilized as a reference in investigating the effectiveness of potential anthrax vaccines produced by Koreans in the Republic of Korea.

The genome sequence of B. anthracis was sequenced, new strategies to combat the disease are being explored. Bacteria have devised a variety of ways to defy detection of our immune system. The primary method for hiding from detection that is employed by every bacteria is molecular camouflage. Modifications to the outer layer render bacteria virtually invisible to the lysozymes. 3 of the modifications are identified, and characterised.

These include (1) N-glycosylation of N-acetyl-muramic acid, (2) O-acetylation of N-acetylmuramic acid and (3) N-deacetylation of N-acetyl-glucosamine. The research conducted over the past few years has been focused on preventing the effects of these modifications. In the process, the mechanism that enzymatically controls polysaccharide deacetylases is being studied which catalyze eliminating an acetyl molecule from N-acetylglucosamine and N acetyl- which are the constituents of the peptidoglycan layers.

Anthrax as a Biological Weapon

Anthrax is considered to be as one of the top bioterrorism risks. In late in 20thCentury, B. anthracis was developed by various nations in their bio weapon (BW) programmes. The autonomous groups have also shown the intention to use B. anthracis to carry out terrorist acts. For instance, as shown in a transcript of March 10, 2007 Department of Defense transcript of the Tribunal Hearing of Khalid Sheikh Muhammad, al Qaeda leadership has expressed interest in and been working to develop anthrax and various other weapons derived from biological sources. In 1993 in 1993, the Japanese popular cult Aum Shinrikyo sprayed aerosols containing B. anthracis multiple times during attempted attack in Tokyo.

Fortunately, the materials was found to be ineffective which meant that no one was affected. In particular, in the month of October 2001 anthrax attacks were committed in the US via mail, when envelopes with B. anthracis-related spores was shipped through the US postal system. (4 were found). The result was 22 cases of anthrax (11 inhalational attacks, 11 cutaneous) 5 individuals who died of inhalational anthrax. In 2009 the FBI ended an investigation on the source of the attacks, concluding the doctor. Bruce Ivins, an Anthrax researcher at US Army Medical Research Institute of Infectious Diseases was responsible for the attack. But Ivins was not charged. Ivins committed suicide before charges could be brought, which meant that the investigation was not prosecuted. Some organizations have challenged the FBI’s decision.

There are a variety of factors that contribute to the concern about the possible use of B. anthracis to create a weapon of biological warfare:

  • B. anthracis are available in microbe bank stores around the globe.
  • B. anthracis has been found to be accessible naturally in endemic regions.
  • There is evidence to suggest that methods for mass production as well as the dispersion of aerosols of anthrax has been devised.
  • The toughness of spores of anthrax within the environment could allow for anthrax aerosol to be more efficient than other agents.
  • Inhalational anthrax that is not treated is a deadly disease with a high mortality rate.
  • The antibiotic resistant strains from B. anthracis are present in nature and can be employed in an intentionally released release.
  • Anthrax was employed in the past as an abio weapon.

A study conducted in 1993 from the Office of Technology Assessment of the US Congress estimated that between 130,000-3 million deaths could result from an explosion of around 100 kgs in air-borne B. anthracis above Washington, DC, making this attack just as deadly as hydrogen bombs.

FAQ on Bacillus anthracis 

Q1. what tests do you suggest to distinguish between bacillus anthracis and bacillus cereus?

Bacillus anthracis and Bacillus cereus can usually be distinguished by standard microbiological methods (e.g., motility, hemolysis, penicillin susceptibility and susceptibility to gamma phage) and PCR.

Q2. which of the following is not a characteristic of bacillus anthracis?

A) Aerobic

B) Gram-positive

C) Forms endospores

D) Found in soil

E) Produces endotoxins

Answer: E

Q3. which of the following forms of anthrax is transmitted by the endospores of bacillus anthracis?

a. cutaneous only

b. cutaneous and inhalation

c. cutaneous, gastrointestinal, and inhalation

d. inhalation and gastrointestinal

Answer: C

Q4. which scientist showed that anthrax was caused by the bacterium, bacillus anthracis?

Scientist Robert Koch studied Bacillus anthracis, the bacterium that causes anthrax. He discovered that the bacteria formed spores and were able to survive for very long periods of time and in many different environments.

Q5. why are antimicrobial drugs of limited usefulness in bacillus anthracis infections?

a. B. anthracis grows so slowly that it is hard to kill using antimicrobial drugs.

b. Most strains of B. anthracis are resistant to a wide variety of antimicrobial drugs.

c. Antimicrobial drugs are unable to neutralize anthrax toxin.

d. Anthrax toxin is absorbed into the bloodstream very quickly.

Answer: C

Q6. which statement reflects the nursing management of pulmonary anthrax (bacillus anthracis)?

Q7. how is bacillus anthracis transmitted?

People get anthrax by Breathing in spores, Eating food or drinking water that is contaminated with spores, or. Getting spores in a cut or scrape in the skin.

Q8. how does bacillus anthracis obtain energy?

Bacillus anthracis is a Gram-positive, rod-shaped bacterium, 1 – 1.2µm in width and 3 – 5µm in length. It lives in soils worldwide at mesophilic temperatures. It can be grown in aerobic or anaerobic conditons (facultative anaerobe) in a medium with essential nutrients, including carbon and nitrogen sources 

Q9. how does bacillus anthracis move?

Anthrax is usually spread in the form of a spore. (A spore is a dormant form that certain bacteria take when they have no food supply. Spores can grow and cause disease when better conditions are present, as in the human body.)

References

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  • Anthrax in Humans and Animals. 4th edition. Geneva: World Health Organization; 2008. Annex 1, Laboratory procedures for diagnosis of anthrax, and isolation and identification of Bacillus anthracis. Available from: https://www.ncbi.nlm.nih.gov/books/NBK310485/
  • Topley WWC (2007). Topley and Wilson’s Microbiology and Microbial Interactions; Bacteriology, 2 Vol. Tenth Edition. John Wiley and Sons Ltd.
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  • Bhatnagar R, Batra S. Anthrax toxin. Crit Rev Microbiol. 2001;27(3):167-200. doi: 10.1080/20014091096738. PMID: 11596878.
  • Bergey, D. H., Whitman, W. B., De, V. P., Garrity, G. M., & Jones, D. (2009). Bergey’s manual of systematic bacteriology: Vol. 3. New York: Springer
  • Anthrax in Humans and Animals. 4th edition. Geneva: World Health Organization; 2008. 2, Etiology and ecology. Available from: https://www.ncbi.nlm.nih.gov/books/NBK310478/
  • Turnbull PCB. Bacillus. In: Baron S, editor. Medical Microbiology. 4th edition. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 15. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7699
  • Zasada, Aleksandra A. “Detection and Identification of Bacillus anthracis: From Conventional to Molecular Microbiology Methods.” Microorganisms vol. 8,1 125. 16 Jan. 2020, doi:10.3390/microorganisms8010125
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  • Keim, Paul et al. “The genome and variation of Bacillus anthracis.” Molecular aspects of medicine vol. 30,6 (2009): 397-405. doi:10.1016/j.mam.2009.08.005
  • Ezzell JW, Welkos SL. The capsule of Bacillus anthracis, a review. J Appl Microbiol. 1999 Aug;87(2):250. doi: 10.1046/j.1365-2672.1999.00881.x. PMID: 10475959.
  • Koehler, Theresa M. “Bacillus anthracis physiology and genetics.” Molecular aspects of medicine vol. 30,6 (2009): 386-96. doi:10.1016/j.mam.2009.07.004
  • Goel, Ajay Kumar. “Anthrax: A disease of biowarfare and public health importance.” World journal of clinical cases vol. 3,1 (2015): 20-33. doi:10.12998/wjcc.v3.i1.20
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