The pathogenesis of Bacillus anthracis
By [Tony Amolo]
Anthrax is a highly contagious disease caused by the bacteria named Bacillus anthracis from the family Bacillaceae. It is the only obligate bacillus pathogen in vertebrates as other Bacillus species are harmless saprophytes. Based on phenotype, it can be classed with Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides as it is difficult to differentiate this group based on their 16S rRNA sequences (10). The genus includes thermophilic, psychrophilic, acidophilic, alkaliphilic, halotolerant, and halophilic, which are capable of growing at temperatures, pH values, and salt concentrations at which few organisms could survive.(4) Named by Hippocrates for the black skin lesions it causes in its cutaneous form, anthrax( Greek: “coal) was well known in the ancient past and is featured in two of the ten plagues of the Old Testament.(1) It generally affects warm blooded animals including humans. Aloys Pollender, who is credited with discovering the disease called the organism “chyllus corpuscles” in the blood spleen, and carbuncle fluid of cows, which had died of the disease (2). Anthrax was extensively studied in the 1870s by Robert Koch and Louis Pasteur. Koch used suspended drop culture methods to trace the complete life cycle of the bacteria and found that the spores formed could remain viable for long period of adverse environments (3). It became a matter of public interest after the bioterrorist attacks in the USA during the autumn of 2001 where five letters containing small quantities of anthrax spores contaminated more than 30,000 people, killed five people and infected 17. (13)
Cell Structure and Metabolism
Bacillus anthracis is an endospore forming aerobic or facultative anaerobic, rod shaped, immobile and capsulated bacteria. It is a gram positive bacteria but in some cultures may turn gram negative with age (4). Bacillus anthracis is 1-1.5micrometer wide by 5-6 micrometer in length. They look like bamboo canes in tissue (13). Many species of its genus exhibits a wide range of abilities that allow them to live in different natural environment. (4) They have the ability to form only one endospore per cell. These spores are usually formed when vegetative cells are deprived of certain nutrient. Bacillus anthracis requires oxygen to sporulate and its spores are resistant to cold, heat, radiation, desiccation and disinfectants.(8) Spores are oval and sporulation occurs within 48hours. Spore formation begins with asymmetric septation then the mother cell engulfs the forespore and protects it with three layers called the coat, cortex and exosporium. The exosporium is a loose-fitting, balloon-like structure enclosing a spore that has been studied to understand the use of anthrax as a weapon. Concerns about the use of Bacillus anthracis spores as a biological weapon have resulted in efforts to understand the interactions between Bacillus anthracis and the cells of the mammalian immune system, and to develop better detectors for these spores. (8) Bacillus anthracis has been successful as a bioweapon because of its tough resistant endospore and its ability to cause lethal inhalational anthrax.
Habitat and Ecology
Bacillus anthracis is mostly found in the agricultural regions of Central and South America, sub-saharan Africa, central and southwestern Asia, southern and eastern Europe, and the Caribbean. It is a soil borne bacteria. It lives best in black steppe soils especially chernozem and kastanozem, rich in organic matter and calcium at pH levels between (7-9). Endemic anthrax areas have been associated with warmer temperatures, higher soil moisture content and topography (15). Most research suggests that germination and multiplication occurs in the host while spore survival occurs in the soil.
Pathogenesis and Virulence factors
Bacillus anthracis depends on two toxic factors from two plasmids:
the pX01, 182kb,which encodes the genes for the edema factor (EF), the lethal factor (LF) and the protective antigen (PA), and the
pX02, 96kb, which encodes the genes for the biosynthesis of the capsule. Bacillus anthracis expresses it pathogenic action mainly through the capsule and the production of a toxic complex consisting of three proteins(EF, LF and PA). (13) The lethal toxin(LT, the combination of PA and LF) and edema toxin (ET, the combination of PA and EF) are sufficient to produce many of the symptoms of anthrax infection.(7) Of the three factors, the PA plays a fundamental role in the toxic action of Bacillus anthracis. Protective antigen (735 amino acids) plays an important role in anthrax intoxication by mediating the attachment and entry of the lethal factor and Edema factor into the cytosol. It contains regions involved in binding to the cell receptor, binding LF and EF, membrane insertion, and translocation of the anthrax toxin. Protective antigen is a major immunogen present in anthrax vaccines(11). The edema factor(89kDa) is a calcium and calmodulin-dependent adenylate cyclase that causes a dramatic increase in cytoplasmic cAMP leading to an imbalance of water homeostasis. The edema toxin may increase host susceptibility to infection by disrupting the cytokine response of monocytes and by suppressing neutrophil functions (11). The lethal factor is a Zn2+ dependent metalloprotease that cleaves major pathways to surface receptors for the transcription of certain genes within the nucleus(12) while the capsule enhances the virulence by inhibiting the phagocytosis of bacillus anthracis. Anthrax toxins provided a mechanism of trans-membrane translocation of proteins, the discovery of plasmids pXO1 and pXO2 which allowed the study of virulence factors at a genetic level(5).
Transmission and Pathology
Bacillus anthracis is not an invasive organism. The most common ways of transmission of the anthrax disease are through micro-wounds, mucous membranes of the mouth, pharynx, gastrointestinal tract, the digestive tract after ingestion of spore contaminated feed or water, skin lesions or abrasions caused by biting flies and the inhalation of dust containing spores. After transmission through one of these route, the spores of Bacillus anthracis are carried from the site of entry to the draining lymph nodes where they begin to multiply rapidly and the produce spores which germinate to produce the virulence factors (13). The pathogenicity of Bacillus anthracis depends on the sensitivity of the host, on the infectious dose(amount of toxin produced), the quality of the capsular coat and the route of penetration (13). Anthrax is not contagious from sick to healthy animals but by the ingestion of spores dispersed into the environment.
Anthrax in Humans
Cutaneous anthrax Cutaneous anthrax is also called malignant pustule. It is usually localized around the face, arms, hands, and neck.It occurs through the contamination of a cut or abrasion that creates an entry for the organism. A primary lesion is formed with a few days and a ring of vesicles develops around the central papule. This dries up to form a black lesion. The lesion develops for 12-14 days leaving a shallow ulcer that heals in 2-3 weeks. Patients show symptoms of headaches, fever of up to 102 degree Fahrenheit, discomfort and even death.
Pulmonary(inhalation) anthrax Also known as woolsorter’s disease, pulmonary anthrax is caused by inhaling the anthrax spores. Spores are phagocytosed by alveolar macrophages and transported to the lymph nodes where they germinate and multiply. Fatal bacteraemia and toxaemia then ensue. (10) During the initial stage, the patient experiences mild fever, malaise, fatigue and cough. The second stage include symptoms like acute dyspnea, cyanosis and profuse perspiration
Gastrointestinal anthrax Also known as splenic fever, gastrointestinal anthrax is extremely rare and occurs mainly in Africa, the Middle East and central and Southern Asia. It is caused by ingestion of insufficiently cooked meat contaminated with spores. Symptoms include diarrhea, ulcer, liver disease and fever. Another rare form of anthrax in man is anthrax meningitis. (6)
Vaccines and Treatment
Anthrax was the first bacterial disease for which effective preventive treatment (prophylaxis) was developed(11). Prophylaxis played a major role in controlling anthrax in animals and protection to individuals from infection. Several vaccines has been produced for the anthrax disease since Louis Pasteur first produced the attenuated anthrax vaccine in 1881 (2). The vaccines used to fight anthrax are composed of spores from weakened strains of Bacillus anthracis. They are classified into two categories namely;
Live attenuated vaccines, capsulated and atoxigenic cap
Live attenuated spore vaccines, not capsulated and toxigenic cap. For example, Sterne and sexually transmitted infection (STI) vaccines. (13)
Other vaccines include non-living vaccines, In vitro protective antigen, Boor and Tresselt vaccine, production in non-proteinaceous media, UK vaccine, American vaccines(Aerobic antigen and Anaerobic antigen) and Russian antigen (6). Special therapy can also be provided to treat anthrax if it is applied immediately to the infected animal or human. Aside from vaccines, antitoxin and antibiotics have been used to treat the disease. Penicillin is used for treatment of susceptible strains of anthrax, with ciprofloxacin and doxycycline as suitable alternatives.(14) Several methods have been adopted to create an improved human vaccine. Recombinant DNA methodology is being used to create live vaccine strains of Bacillus anthracis. The aim of the anthrax vaccine create a new vaccine that is safe, non reactogenic, efficacious against all disease and requires a minimum number of inoculations to achieve maximum long lasting immunity (11). The most challenging aspect of evaluating the efficacy of anthrax vaccine is making a logical assessment in humans. Animals can be used as a model but the process can be dangerous.
Application to Bioterrorism
Bacillus anthracis is one of the few pathogenic bacteria that can be used as for bioterrorism. It is most likely to be used because it spores can be produced in the lab,last for a long time in the environment and can be found easily in nature. Also because of its small size, it can be placed in food and letters like in 2001 when powdered anthrax was mailed to the U.S postal office. Twenty-two cases of anthrax infections were confirmed. Half of these cases involved cutaneous anthrax and no death resulted. The remaining eleven were identified as inhalational anthrax and resulted in five deaths. A global awareness should be created to reject the development of such weapons and early detection of the disease.