Bacillus spp.

large Gram-positive rods that chain

spore-formers

found as saprophytes in soil, water, and vegetation

34 different species

some are aerobes; others facultative anaerobes

some important as producers of antibiotics, e.g. bacitracin and polymyxin

Only 2 species are pathogenic to humans:

**B. anthracis, the etiologic agent of anthrax

B. cereus, may cause food poisoning

 

Bacillus anthracis

anthrax bacillus

nonmotile

facultative anaerobe

4 to 10 µm in length and 1 to 1.5 µm in width- BIG!

grows at temps 15-40°C

extracellular pathogen

colonies - rough in texture

Spores - produced during conditions that are unfavorable for growth of vegetative cells: in in vitro culture, in the soil, or in the tissues of dead animals. Sporulation by B. anthracis requires oxygen. The spores may remain viable for years. Spore is oval and central (endospore)

Spores are not seen in the blood or tissues of living animals. In vivo, B. anthracis appears as non-sporeforming rods in short chains.

 

Pathogenesis of anthrax (zoonosis)

Primarily a disease of herbivorous animals (sheep, cattle, goats, and horses). Acquired through ingestion or inhalation of spores while feeding. Animal disease is severe; septicemia; mortality ~80%. Bacilli shed from infected animal in large numbers. During accept phase: hemorrhaging from small blood vessels producing bloody exudations (characteristic) carrying large numbers of bacilli (109/ mL of blood).

Humans - accidental hosts; acquired by contact with diseased animals, their hides or hair. Anthrax is considered an occupational hazard for those working with livestock (agricultural anthrax - farmers, vets, and butchers) or where animal hides are processed (industrial anthrax). No known case of human-to-human transmission.

 

a. Cutaneous anthrax - most common form (95%) of the human disease. Organisms gain entrance to the subcutaneous tissue through abraded skin or mucosa. The skin lesion is painless and appears as a dark necrotic area surrounded by a rim of edema. The spores germinate in the tissue, and vegetative cells produce an exotoxin. Often, limited lesion heals spontaneously. However, if the bacilli reach the lymph nodes and bloodstream (small % of cases), fatal septicemia may develop.

The pathophysiology of toxin and infection are not well understood. The primary site of action of the toxin is not known for certain but appears to be the CNS. Symptoms: cardiac failure, increased vascular permeability, shock, and respiratory failure.

2000 cases/yr world-wide, 224 US 1944-94, 10,000 case in Zimbabwe 1979-85

 

 

 

b. Gastrointestinal anthrax - uncommon in the US, but it may occur in underdeveloped countries where poorly cooked meats from contaminated animals are sold. The toxin produced in the intestinal tract forms a necrotic lesion in the ileum or cecum. Symptoms: nausea, vomiting, and diarrhea. Hemorrhagic inflammation of the small intestine may occur with bowel perforation. Fatality rates are high for this condition. Two distinct syndromes: oral-pharyngeal and abdominal.

24 cases oral-pharyngeal from contam buffalo meat Thailand 1982, 14 cases of mix oral-pharyngeal and abdominal in Thailand 1987

In cutaneous and gastrointestinal anthrax: low level germination of spores at primary site -> local edema & necrosis -> spores phagocytosed by Mphages and germinate -> migrate to lymph nodes -> veg cells grow -> regional hemorrhagic lymphadentitis -> septicemia through blood and lymph spread, exotoxins produced. Meningitis occurs in small number of cases.

c. "Woolsorter’s" disease - inhalation of anthrax spores resulting in pulmonary anthrax. The inhaled spores (from exposure to animal hair or hides) settle in the respiratory tract, producing pneumonia with local hemorrhage and edema.

spores phagocytosed by Mphages -> migrate to lymph nodes and germinate (up to 60d later) -> veg cells grow -> peribronchial hemorrhagic lymphadentitis -> blockage of pulmonary lymphatic drainage -> pulmonary edema

Septicemia may occur leading to meningitis. Because the disease is rarely recognized before bacteremia has developed, it progresses rapidly and is often fatal. For humans LD50 = 2500-55000 inhaled anthrax spores. anthrax pneumonia: misleading, not typical bronchopneumonia, more hemorrhagic

 

18 reported cases 1900-1978, majority in specialty groups, none since 1978 until case in Florida 2001.

Pulmonary anthrax is considered as a means of biologic warfare, which has now been outlawed by international treaty

Death – septicemia, toxemia or pulmonary complications within 1 to 7 days after exposure.

Epidemiology

Naturally occurring (non-biowarfare) anthrax - rare in US; more common in Iran, Turkey, Pakistan, Sudan (endemic).

 

Virulence determinants - both plasmid-encoded

1. Capsule: in the presence of elevated concentration of CO2, B anthracis colonies become mucoid due to elaboration of a capsule - single capsule type: high-molecular weight polypeptide of poly-D-glutamic acid. Genes encoding proteins involved in capsule expression are carried on a ~97-kb plasmid (pX02).

B. anthracis is encapsulated during growth in the infected animal.

The capsule is antiphagocytic, but it is a poor immunogen. Its importance in bacterial virulence is believed to lie in the establishment of infection; nonencapsulated mutants are avirulent. The terminal phases of the disease is attributed to in vivo toxin production.

 

2. Toxin - genes involved in exotoxin secretion are carried on a ~184-kb plasmid (pX01). The exotoxin is a complex of three distinct proteins. Together they act synergistically to produce the characteristic systemic effects of anthrax. Individually, the proteins are nontoxic.

 

a. protective antigen (PA) - 83-kDa protein; binds to a membrane receptor on host cells. Host proteases in the blood and on the eukaryotic cell surface activate protective antigen by cleaving a 20-kDa segment, exposing a binding site for lethal factor and edema factor. The complex is then internalized into eukaryotic cells by endocytosis. Acidification of the endosomes causes translocation of LF or EF to the cytosol.

b. edema factor (EF). This is an inactive form of the enzyme adenylate cyclase. Its function is to convert ATP to cAMP. In the cytoplasm, EF interacts with the eukaryotic cofactor calmodulin and Ca++ to become an active adenyl cyclase. The production of cAMP causes mammalian cells to over secrete fluid resulting in edema. Local extracellular edema is a feature of cutaneous anthrax.

c. Lethal factor (LF) - LF is a metalloprotease that causes hyperinflammatory condition in Mphages. LF is a protease that cleaves and inactivates Mitogen-activated protein kinase kinase (MAPKK), a cellular enzyme important in a cell’s signaling pathways. MAPKK regulates the activity of other cellular molecules by attaching phosphate groups to them. LF cleaves the amino terminus of this protein, inactivating the MAPKK and resulting in the inhibition of the MAPK signal transduction pathway (helps control cell growth, embryonic development, and maturation of oocytes into eggs). Activation of the oxidative burst pathway and release of oxygen intermediates and production of proinflammatory cytokines (TNF-alpha and IL-1beta)

PA + LF causes lethality in rats, mice and guinea pigs

PA + EF injected intradermally cause local edema in the skin of rabbits or guinea pigs.

The anthrax toxin complex is also toxic for phagocytic cells of some mammalian species.

 

Lab diagnosis

B. anthracis can be cultured from soil, cutaneous lesion, the respiratory tract, or blood. However, B. anthracis must be differentiated from other sporeforming bacilli that are common contaminants.

B. anthracis is the only species that is nonmotile, nonhemolytic, sensitive to penicillin (others are not), and produces a capsule under elevated CO2 conditions, with a central to terminal spores.

Other detection methods: serologic tests - capsule and exotoxin ELISA’s for past infection or vaccination, immunologic tests unreliable, PCR used for amplification of specific genes on chromosome or plasmids under development.

 

Immunity

Recovery from infection provides immunity to reinfection.

Antibodies elicited against killed bacteria are not protective. Protective abs are directed against the exotoxin complex. A toxoid prepared from a fraction of the exotoxin (protective antigen) is safe as a vaccine for humans.

The currently licensed human anthrax vaccine (1970) is a cell-free supernatant material (primarily protective antigen) from a toxigenic, nonencapsulated attenuated strain of B. anthracis. The vaccine is injected SC (6 doses/18 mo) and may cause localized, painful reaction. Booster doses are required yearly thereafter (mandated for all US military, active and reserve-duty personnel). In countries of former Soviet Union a human live attenuated vaccine is used.

Anthrax vaccines for livestock are spores of a live attenuated vaccine strain that is nonencapsulated. Administered yearly; not currently administered in the U.S.

 

Treatment and Prevention

Penicillin or doxycycline is the drug of choice. Alternatives are streptomycin, tetracycline, and erythromycin. Inhalation anthrax, ciprofloxicin as initial therapy, PenG and Dox opitmal therapy if shown to be susceptible. Early treatment is important because antibiotics are not effective against the toxin. Abic treatment does not allow for immunity to develop thus treatment is for 60d (spores).

Anthrax still causes heavy losses of livestock worldwide. The most effective control program is vaccination of cattle, cremation of infected animals, and restricted movement of livestock.

 

BIOLOGIC WEAPON

Developed by a number of nations:

used by Japansese army in Manchuria in 1940s

1979 Sverdlovsk, Russia – outbreak in humans due to accidental release, 5000 exposed - 79 infected/68 deaths

Aum Shinrikyo released anthrax and botulinum aerosols in Japan 1995

Biologic warfare: inhalation of aerosolized spores leading to respiratory failure and death in <1wk. Aerosol is odorless and invisible, travel kilometers before disseminating

Biologic attack: post exposure vaccination is recommended with antibiotic administration (residual spores). Inhalation anthrax, early abic admin is essential due to rapid progression of disease.

A pen and tet-class resistant strain developed by Russians, no accounts of quinolone-resistance

 

Bacillus cereus

 

Present on many grains, vegetables and dairy products) produces two enterotoxins elaborated by germinating spores. They are responsible for 2 self-limiting clinical entities.

Cooking kills vegetative bacteria but heat-resistant spores survive. If food is not refrigerated, spores germinate and bacteria multiply, elaborating toxin.

 

a. typical gastroenteritis characterized by diarrhea and abdominal pain for <24 h. Occurs 8 to 16 h after ingestion of contaminated food (meat, vegetables). It is a heat-labile adenylate cyclase enzyme. Sel

b. emetic type caused by heat stable enterotoxin; occurs 1 to 5 h after ingestion of contaminated food (e.g. fried-rice dishes). ?Mechanism of action.

 

B. cereus and other nonpathogenic species may cause serious infection in-patients with impaired host defenses. Non-anthrax species are resistant to penicillin. Treat with clinda, aminoglycosides, vanco, tet, em, etc.