Bacillus species: Introduction, Classification and about Bacillus anthracis

Bacillus species: Introduction, Classification and About Bacillus anthracis and cereus

Introduction Bacillus species

Bacillus species on Chocolate agar as shown above image. It is ubiquitous in nature and remains both free-living (non-parasitic) species, and two parasitic pathogenic species, B. anthracis causes anthrax; and B. cereus causes food poisoning and thus these two Bacillus species are medically significant. The word, Bacillus is Latin which means stick. Bacillus is a genus of Gram-positive,

rod-shaped bacteria, a member of the phylum Firmicutes, with 266 named species. Bacillus species are obligate aerobes as well as facultative anaerobes and catalase test positive. Bacillus can reduce themselves to oval endospores and they can remain in this dormant state for years. They are generally motile with peritrichous flagella. The genus includes psychrophilic, mesophilic, and thermophilic species. The maximum temperatures for vegetative growth ranging from about 25°C to above 75 °C and the minimum from about 5 °C to 45°C. Their salt tolerance varies from less than 2 % to 25 % NaCl. Their spores are ubiquitous, being found in the soil, dust, water, and air, and constitute the commonest contaminants in bacteriological culture media.

Scientific classification

Domain: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Bacillaceae
Genus: Bacillus
Species: B. anthracis
Binomial name: Bacillus anthracis
Bacillus cereus

Bacillus anthracis

Robert Koch isolated in pure culture and observed the spores in 1876. Louis Pasteur in 1881 prepared for the attenuated vaccine. Primarily a disease of domesticated and wild animals – Herbivores such as sheep, cows, horses, goats. Natural reservoir is soil – Does not depend on an animal reservoir making it hard to eradicate – Cannot be regularly cultivated from soils where there is an absence of endemic anthrax – Occurs sporadically throughout the US – South Dakota, Arkansas, Texas, Louisiana, Mississippi, California recognized endemic areas. Anthrax was for long feared as a potential tool in biological warfare. This fear became an actual fact in 2001, when anthrax in the form of weapons-grade spores having enhanced dispersibility and virulence was sent by mail to various destinations in the USA, causing disease and death in many persons.

Morphology of Bacillus anthracis

The anthrax bacillus is one of the largest pathogenic bacteria, measuring 3-10 µm x 1-1.6 µm. In tissues, it is found singly, in pairs, or in short chains, the entire chain is surrounded by a capsule. The capsule is polypeptide in nature. Capsules are not formed under ordinary conditions of culture but only if the media contain added bicarbonate or are incubated under 10-25 % CO₂. If grown in media containing serum, albumin, charcoal, or starch, capsule formation may occur in the absence of CO₂. In cultures, the bacilli are arranged end to end in long chains like a ‘bamboo stick’ appearance.
Spores are formed in culture or in the soil but never in the animal body during life. Spores are central or sub-terminal, elliptical or oval in shape, and are of the same width as the bacillary body so that they do not cause bulging of the vegetative cell. The anthrax bacillus is Gram-positive and non-acid fast.
The spores do not stain by ordinary methods but can be stained deferentially by special techniques When stained with Sudan black B, fat globules may be made out within the bacilli. M”Fadyean’s reaction: When blood films containing anthrax bacilli are stained with polychrome methylene blue for a few seconds and examined under the microscope, amorphous purplish material is noticed around the bacilli. This represents the capsular material and is characteristic of the anthrax bacillus. This is called the M”Fadyean’s reaction is the presumptive diagnosis of anthrax in animals. The anthrax bacillus is non-motile, unlike most other members of this genus.

Culture Characteristics of Bacillus anthracis

It is an aerobe, and facultative anaerobe, with a temperature range for growth of 12-45 °C (optimum 35-37 °C). Good growth occurs on ordinary media. On agar plates, irregularly round colonies are formed, 2-3 mm in diameter, raised, dull, opaque, greyish white, with a frosted glass appearance. Under the low power microscope, the edge of the colony is composed of long, interlacing chains of bacilli, resembling locks of matted hair. This is called the ‘Medusa head appearance’

On blood agar, the colonies are nonhemolytic. In broth, growth occurs as a floccular deposit. When B. anthracis is grown on the surface of a solid medium containing 0.05-0.50 units of penicillin/ml, in 3-6 hours the cells become large, spherical, and occur in chains on the surface of the agar, resembling a string of pearls. This ‘string of pearls reaction’ differentiates clearly B anthracis from B cereus and other aerobic spore bearers. A selective medium (PLET medium), consisting of polymyxin, lysozyme, ethylene diamine tetraacetic acid (EDTA) and thallous acetate added to heart infusion agar has been devised to isolate B. anthracis from mixtures containing other spore-bearing bacilli.

Biochemical test

Glucose, maltose, and sucrose are fermented producing acid but no gas. Nitrates are reduced to nitrites. Catalase is formed. On gelatin stab culture, a characteristic ‘inverted fir tree’ appearance is seen, with slow liquefaction commencing from the top.

Resistance

The vegetative bacilli are not particularly resistant and are destroyed at 60°C in 30 minutes. In the carcasses of animals that have died of anthrax, the bacilli remain viable in the bone marrow for a week and in the skin for two weeks. The spores are highly resistant to physical and chemical agents. They have been isolated from naturally infected soil after as long as 60 years. They resist dry heat at 140°C for 1-3 hours and boiling for 10 minutes. They survive in 5 % phenol for weeks. HgCl2 in a 1/1000 solution may fail to kill anthrax spores in less than 70 hours. 4% potassium permanganate kills them in 15 minutes. Destruction of the spores in animal products imported into non-endemic countries is achieved by ‘Duckering’ in which 2% formaldehyde solution is used at 30-40 °C for 20 minutes for disinfection of wool and as 0.25 % at 60°C for six hours for animal hair and bristles. The anthrax bacillus is susceptible to sulphonamides, penicillin, erythromycin, streptomycin, tetracycline, and chloramphenicol. Occasional strains resistant to penicillin are encountered.

Pathogenicity

In nature, anthrax is primarily a disease of cattle and sheep, and less often of horses and swine but experimentally most animals are susceptible to a greater or lesser degree. Rabbits, guinea pigs, and mice are susceptible. Following the subcutaneous inoculation of a culture into a guinea pig, the animal dies in 24 to 72 hours showing a local, gelatinous, hemorrhagic edema at the site of inoculation, extensive subcutaneous congestion and characteristically, an enlarged, dark red, friable spleen. The bacilli are found in large numbers in the local lesion, heart blood, and spleen (> 10⁸bacilli/ ml).

Virulence Factors

Two virulence factors have been identified- Capsular polypeptide and Anthrax toxin. The capsular polypeptide aids virulence by inhibiting phagocytosis. Loss of the plasmid (pX 02) which controls capsule production leads to loss of virulence. This is how the live attenuated anthrax spore vaccine was obtained. The anthrax toxin was identified by the finding that the injection of sterile plasma of guinea pigs dying of anthrax into healthy guinea pigs killed them and that death could be prevented by the immune serum. The toxin is a complex of three fractions: the edema factor (OF or Factor I), the protective antigen factor (PA or Factor II), and the lethal factor (LF or Factor III). They are not toxic individually but the whole complex produces local edema and generalized shock. The three factors have been characterized and cloned. PA is the fraction that binds to the receptors on the target cell surface, and in turn provides attachment sites for OF or LF, facilitating their entry into the cell.
The antibody to PA is protective because it blocks the first step in toxin activity, namely, its binding to target cells. OF is an adenyl cyclase that is activated only inside the target cells, leading to intracellular accumulation of cyclic AMP. This is believed to be responsible for the edema and other biological effects of the toxin. Entry of LF into the target cell causes cell death but the mechanism of action is not known Loss of the plasmid (pX 01) which encodes the F toxin renders the strain avirulent. This is believed to have been the basis for the original anthrax vaccine developed by Pasteur. The avirulent Sterne vaccine strain is devoid of the plasmid coding for the capsular polysaccharide. Anthrax is a zoonosis. Animals are infected by ingestion of the spores present in the soil. Direct spread from animal to animal is rare. The disease is generally fatal septicemia but may sometimes be localized, resembling the cutaneous disease in human beings. Infected animals shed in the discharges from the mouth, nose, and rectum, large numbers of bacilli, which sporulate in soil and remain as the source of infection. Human anthrax is contracted from animals, directly or indirectly. The disease maybe

cutaneous
pulmonary
intestinal, all types leading to fatal septicemia or meningitis.

Cutaneous Anthrax

This follows the entry of the infection through the skin. The face, neck, hands, arms, and back are the usual sites. The lesion starts as a papule 1- 3 days after infection and becomes vesicular, containing fluid that may be clear or bloodstained. The whole area is congested and edematous, and several satellite lesions filled with serum or yellow fluid are arranged around a central necrotic lesion which is covered by a black eschar (The name anthrax, which means coal, comes from the black color of the eschar). The lesion is called a malignant pustule. The disease used to be common in dock workers carrying loads of hides and skins on their bare backs and hence was known as the hide porter’s disease. Cutaneous anthrax generally resolves spontaneously, but 10-20 percent of untreated patients may develop fatal septicemia or meningitis.

Pulmonary Anthrax

This is called the wool sorter’s disease because it used to be common in workers in wool factories, due to the Inhalation of dust from infected wool. This is hemorrhagic pneumonia with a high fatality rate. Hemorrhagic meningitis may occur as a complication.

Intestinal Anthrax

This is rare and occurs mainly in primitive communities that eat the carcasses of animals dying of anthrax. Violent enteritis with bloody diarrhea occurs, with high case fatality.

Human Anthrax

Human anthrax may be industrial or non-industrial (agricultural). The former is found in workers in industries such as meatpacking or wool factories. Non-industrial anthrax is often an occupational disease in those who associate frequently with animals, such as veterinarians, butchers, and farmers. It may also be found in the general population. Cutaneous anthrax used to be caused by shaving brushes made with animal hair. Stomoxys calcitrans and other biting insects may occasionally transmit infection mechanically.

Laboratory Diagnosis

Anthrax may be diagnosed by:

Microscopy,
Culture,
Animal inoculation and

Serological demonstration of the anthrax antigen in infected tissues. Antibodies to the organism can be demonstrated by gel diffusion, complement fixation, antigen-coated tanned red cell agglutination, and ELISA techniques. When an animal is suspected to have died of anthrax, an autopsy is not permissible, as the split blood will lead to contamination of the soil. An ear may be cut off from the carcass and sent to the laboratory. Alternatively, swabs soaked in blood or several blood smears may be sent. The demonstration of Gram-positive bacilli with the morphology of anthrax bacilli and a positive M’Fadyean reaction will enable a presumptive diagnosis to be made. Immunofluorescent microscopy can confirm the identification.

n After the bio-terrorism experience in the USA in 2001, the CDC has prepared guidelines for the identification of anthrax bacillus. Any large Gram-positive bacillus with the general morphology and cultural features of anthrax-non-motile, non-hemolytic on blood agar and catalase positive-can be given a presumptive report of anthrax. For initial confirmation, lysis by gamma phage and DFA for capsule-specific staining and for polysaccharide cell wall antigen are sufficient. For further confirmation, PCR for anthrax bacillus-specific chromosomal markers can be done. For epidemiological studies and strain characterization, MLVA or AFLP can be used.

Prophylaxis

Prevention of human anthrax is mainly by general methods such as improvement of factory hygiene and proper sterilization of animal products like hides and wool. Carcasses of animals suspected to have died of anthrax are buried deep in quicklime or cremated to prevent soil contamination. Prevention of anthrax in animals is aided by active immunization. The original Pasteur’s anthrax vaccine is of great historical importance. It was Pasteur’s convincing demonstration of the protective effect of his anthrax vaccine in a public experiment at Pouilly- le- Fort in 1881 that marked the beginning of scientific immunoprophylaxis. Pasteur’s vaccine was the anthrax bacillus attenuated by growth at 42-43 °C. The spore is the common infective form in nature, vaccines consisting of spores of attenuated strains were developed. The Sterne vaccine contained spores of a non-encapsulated avirulent mutant strain. The Mazucchi vaccine contained spores of stable attenuated Carbazoo strain in 2 % saponin. The spore vaccines have been used extensively in animals with good results. They give protection for a year following a single injection. They are not considered safe for human use, though they have been used for human immunization in Russia. Alum precipitated toxoid prepared from the protective antigen has been shown to be a safe and effective vaccine for human use It has been used in persons occupationally exposed to anthrax infection. Three doses are given intramuscularly at intervals of six weeks between first and second, and six months between second and third doses induce good immunity, which can be reinforced if necessary with annual booster injections.

Treatment

Antibiotic therapy is effective in human cases but rarely succeeds in animals as therapy is not started sufficiently early. Antibiotics have no effect on the toxin once it is formed. Penicillin and streptomycin are no longer used for treatment. They have been replaced by doxycycline and ciprofloxacin, which are effective in prophylaxis and treatment.

Bacillus cereus

B. cereus has become an important cause of food poisoning. It is widely distributed in nature and may be readily isolated from soil, vegetables, and a wide variety of foods including milk, cereals, spices, meat, and poultry. B cereus is generally motile but non-motile strains may occur. It resembles B anthracis except that it is not encapsulated and does not react with anthrax fluorescent antibody conjugate. It produces two patterns of food-borne disease. One is associated with a wide range of foods including cooked meat and vegetables. It is characterized by diarrhea and abdominal pain, 8-16 hours after ingestion of contaminated foods. Vomiting is rare. The second type is associated almost exclusively with the consumption of cooked rice, usually fried rice from Chinese restaurants. The illness is characterized by acute nausea and vomiting 1-5 hours after the meal. Diarrhea is not common. B cereus is present in large numbers in the cooked rice and fecal samples from these patients. Both types of illness are mild and self-limited, requiring no specific treatment. Strains causing the emetic type of disease produce a toxin that causes vomiting when fed to Rhesus monkeys, resembling staphylococcal enterotoxin. The emetic toxin was produced only when B. cereus was grown in rice but not in other media. A special mannitol-egg yolk-phenol red- polymyxin agar (MYPA) medium is useful in isolating B. cereus from feces and other sources. It produces lecithinase and ferments glucose but not mannitol.

Anthrocoid Bacilli

A large number and variety of nonpathogenic aerobic spore-bearing bacilli appearing as common contaminants in cultures and having a general resemblance to the anthrax bacilli have been collectively called pseudoanthrax or anthracoid bacilli. Many members of the genus Bacillus, other than the anthrax bacillus, have occasionally caused human infections. It has also been associated with septicemia, meningitis, endocarditis, pneumonia, wound infections, and other suppurative lesions, particularly as an opportunist pathogen. B. subtilis, B. licheniformis, and a few other species have also been occasionally isolated from such lesions.

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