Вacillus Anthracis(causative agent of anthrax)

▶The History of Discovery

The bacterial origin of anthrax was primarily noted by A. Pollander (Germany) in 1849, by K. Davaine (France) in 1850, and by F. Brauell (Russia) in 1854.

The first isolation of anthrax agent was fulfilled by R. Koch in 1876.

In Russia the disease was named as Siberian sore owing to the large epidemic of 1786-1788 in the Urals, described by S. Andreyevsky.

In Germany the infection is known as spleen fever.

▶Classification of Bacillae

Bacillus genus pertains to the same name family Bacillaceae.

Most members of the genus are saprophytic organisms prevalent in soil, water, air, and on vegetations, such as Bacillus cereus and Bacillus subtilis. These bacteria may occasionally produce disease in immunocompromised persons (e.g., meningitis, endocarditis, acute gastroenteritis, etc.)

В. anthracis, which causes anthrax, is the principal pathogen of the genus.

▶Structure and properties of B. anthracis

  • Morphology

B. anthracis are viewed as the large gram-positive rods (1-1.5 μm in breadth and 4-10 μm in length). The microbials are nonmotile, encapsulated, and arranged in chains (streptobacilli).

In stained smears the ends of the bacilli appear to be sharply cut across, resembling bamboo canes. Peptide capsule is usually evident in samples from infected tissues.

The bacilli produce oval subterminal spores that not exceed the width of bacterial cell. Spores are formed only in the presence of oxygen.

  • Cultivation

B. anthracis grows well in ordinary media and sheep blood agar at 37°C, usually forming nonhemolytic rough colonies (R forms) after overnight incubation.

The round colonies are usually flat or slightly convex with irregular edges, sometimes curly tailing edges are observed. It was historically indicated that they resemble the “head of a medusa” or “lion mane.”

The smooth S forms display low virulence or are completely avirulent.

When grown on penicillin-containing meat-peptone agar, the bacilli are transformed into globules that become arranged as a necklace (“pearl necklace”).

Broth cultures of the anthrax bacilli produce flocculent growth as “cotton wool” near the bottom of the tube.

Growth of B. anthracis in gelatin stabs with substrate liquefaction resembles an inverted “fir tree”.

  • Biochemical properties

The anthrax bacilli are aerobic and facultatively anaerobic. They have potent and versatile biochemical activity.

B. anthracis expresses various enzymes – peroxidase, catalase, lipase, amylase. Bacteria utilize proteins producing ammonia and hydrogen sulfide. In addition, anthrax bacilli liquefy gelatin and cause late liquefaction of coagulated serum. They slowly reduce nitrates to nitrites and coagulate milk.

B. anthracis ferments glucose, maltose, sucrose, dextrin, etc. with acid production.

  • Antigenic structure

The anthrax bacilli carry capsular protein and thermoresistant polysaccharide cell wall antigen.

The polysaccharide antigen remains stable for the long period of time in tissues obtained from animal carcasses. The presence of this antigen in raw materials is determined by Ascoli’s thermoprecipitin test – a boiled B. anthracis extract containing thermoresistant Ag yields a precipitin reaction with the specific serum.

The capsular antigen is composed of poly-D-glutamic acid.

B. anthracis produces also a special antigenic fraction, referred to as protective antigen. This antigen is a thermolabile protein with marked immunogenic activity. It takes an active part in metabolism of microbial toxins.

  • Virulence factors

The virulence factors of B. anthracis include two exotoxins and antiphagocytic polypeptide capsule. The loss of the capsule abolishes the virulence of bacteria.

The genes encoding virulence factors of B. anthracis are located in two separate plasmids.

The anthrax toxins are composed of three proteins: PA (protective antigen), and EF (edema factor) or LF (lethal factor).

Protective antigen plays a role of receptor and transport subunit for both microbial toxins. The PA molecule attachs to specific receptors on the host cell membrane. PA is further cleaved by a cellular protease, producing a PA fragment that functions as a specific receptor for edema factor (EF) and lethal factor (LF).

Edema factor is an adenylate cyclase; after binding to PA it forms a toxin known as edema toxin. It suppresses the activity of macrophages and increases vascular permeability resulting in tissue edema.

Lethal factor coupled with PA creates cytotoxic lethal toxin, which is the major virulence factor of B. anthracis.

LF itself is zinc-containing metalloprotease that with high specificity destroys key intracellular regulatory enzyme, namely kinase of mitogen-activated protein kinase (MAPK kinase).

Inactivation of MAPK kinase by lethal toxin profoundly disorganizes intracellular metabolism eventually resulting in cell death.

Both lethal and edema toxins are delivered into the host cell via receptor-mediated endocytosis. Initially confined within endosome, LF and EF cross endosomal membrane and enter the cytoplasm of affected cell through the channel made by protective antigen subunit.

  • Resistance

The intrinsic toughness of B. anthracis spores is outstanding: they may survive in soil for decades (at least more than 50 years). They are grossly more resistant to disinfectants than the vegetative cells. The vegetative bacteria are killed in 15 minutes at 60°C and in 1-2 minutes at 100°C. The spores are thermostable, and withstand boiling for 15-20 minutes or autoclaving at 110°С for 5-10 minutes. They are gradually destroyed by 1% formaldehyde and 10% sodium hydroxide within 2 hours.

▶Epidemiology, Pathogenesis and Clinical Findings in Anthrax

Anthrax is a typical zoonosis. It is enzootic in many parts of the world. The potential source of infection is a vast number of wild and domestic animals (e.g., cattle).

Herbivores become infected with anthrax by grazing in pastures that are contaminated with spores. The animal infection results in bacterial propagation that leads to environmental contamination with vegetative microbial cells. They subsequently sporulate and persist in the soil for 50 years and even more. Animal carcasses are highly infectious. Biting flies can become vectors for the spread of anthrax.

As the environmental conditions play a substantial role in preservation and spread of anthrax germs, the infection caused by B. anthracis is referred to as sapronosis.

Contact with animals (butchering, skinning, or exposure to hides or wool), and consumption of contaminated meat are the risk factors for infection transmission to humans. The incidence of inhalation anthrax is considerably reduced by decontamination procedures for wool and hair.

Depending on the primary portal of entry, anthrax cases demonstrate highly variable clinical manifestations.

In cutaneous anthrax, spores are introduced into the skin by direct contact. Germination occurs within hours, and vegetative cells produce anthrax toxin. The disease usually develops within 1-7 days after entry.

Primary papular-vesicular skin lesion is next changed by ulceration with formation of blackened necrotic eschar or anthrax carbuncle (malignant pustule). This lesion is usually painless. A regional lymphadenitis is commonly observed in these patients. Eventually eschar dries, loosens, and separates; spontaneous healing occurs in 80 to 90% of untreated cases. Bacterial dissemination may lead to systemic infection with high fever and possible lethal outcome.

In case of inhalation anthrax (wool-sorter’s disease), the spores are aerosolized and enter the alveoli of the lungs. The incubation period in inhalation anthrax may last up to 6 weeks.

The spores are ingested by alveolar macrophages and begin to germinate moving to mediastinal lymph nodes. It results in hemorrhagic mediastinitis and massive B. anthracis bacteremia, accompanied by secondary pneumonia. Meningitis may also occur.

The disease manifests by fever, tachypnea, and hypoxia accompanied by hypotension. Severe respiratory distress syndrome leads to lethal outcome within 24 h of the primary phase of infection.

There is no individual human-to-human transmission of inhalation anthrax; nevertheless, this form of disease might be contracted as the result of potential bioterrorist attack due to the high stability of microbial spores.

Accidental gastrointestinal anthrax arises under the ingestion of contaminated meat that was not thoroughly cooked. The course of the disease is severe, fatality is high.

B. anthracis bacteremia occurs in all three forms of human anthrax and it is observed in literally all death cases. Cutaneous anthrax is the most frequent form of disease (95%), next is inhalation anthrax (5%). Gastrointestinal anthrax is extremely rare and may be seen in less than 1% of all clinical cases.

Due to its evident threat to personal and public health, B. anthracis is also placed into Tier 1 of US list of “Biological Select Agents or Toxins” comprising the most dangerous microbial pathogens. Bacillus anthracis ranks high in the list of potential agents of bioterrorist attacks.

▶Laboratory Diagnosis of Anthrax

Depending on the type of infection, B. anthracis may be isolated from various samples: cutaneous lesions, respiratory specimens, stool or other gastrointestinal excretions, blood or cerebrospinal fluid.

The microscopy of Gram-stained smears reveals the presence of characteristic capsulated bacilli, arranged in chains that permits a preliminary diagnosis.

Also anthrax bacilli can be identified by immunofluorescence assay.

Nucleic acids of B. anthracis are determined by PCR.

For isolation of the pure culture the specimens are inoculated into meat-peptone agar, meat-peptone broth and blood agar (the latter yields non-hemolytic colonies). The isolated culture is differentiated from other bacteria by its morphological, biochemical and antigenic properties.

Laboratory animals (mice, guinea pigs and rabbits) are inoculated directly by pathogenic material or by isolated culture. As an example, B. anthracis causes the death of mice in 24-48 hours after inoculation. Microscopical examination of smears made from blood and internal organs reveals anthrax bacilli, which are surrounded by a capsule.

Post-mortem sections as well as leather and hair used as raw materials are examined by thermoprecipitin reaction (Ascoli’s test) to detect anthrax antigens.

Bacterial phage typing is a valuable test, as the specific bacteriophage causes the lysis of pathogenic culture.

In serological diagnosis various kinds of ELISA are developed to determine antibodies against bacterial toxins, capsular and spore-derived antigens. Acute and convalescent sera obtained 3-4 weeks afterwards should be tested. A positive result is a fourfold rise of specific antibody levels.

▶Treatment and Prophylaxis of Anthrax

Penicillin is the drug of choice for treatment of anthrax, but it must be started early. Macrolides, fluoroquinolones (e.g., ciprofloxacin), and vancomycin are also active against these bacteria. Treatment may also include passive immunization with anthrax antitoxic immune globulin.

For specific prophylaxis live (attenuated) vaccine containing spores of non-capsulated B. anthracis vaccine strain is used in many countries to immunize herbivores and groups of humans with high occupational risk of infection.

The vaccine is harmless, but with some side effects; it produces the immunity quite rapidly (in 48 hours) and for a period of over a year. It is inoculated in a single dose.

Another anthrax vaccine is an aluminum hydroxide-precipitated protective antigen.

General measures of anthrax control are carried out in tight cooperation with veterinary workers. These measures are aimed for timely recognition, isolation, and treatment of sick animals. They also include thorough disinfection of premises for livestock, affected territory and all the objects, followed by ploughing the pastures. Carcasses of animals died of anthrax must be burnt or buried in specially assigned areas.