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Aerobic Spore Forming BacilliAerobic Spore Forming BacilliClassification Most members are saprophytic organisms prevalent in soil, water and

air and on vegetation Some are insect pathogensGenus: bacillus – large aerobic, gram +ve rods in chainsSpecies: Bacillus anthrax – principal pathogen, a major agent of bioterrorism

and biologic warfare, causes anthrax Bacillus cereus – cause food poisoning, occasionally eye or other

localized infection Bacillus subtilis B. thuringiensis, B. popilliae, B. sphaericus, B larvae and B.

lentimorbus – pathogens for insects, commerc. insecticides

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Bacillus SpeciesBacillus SpeciesMorphology and Identification

Typical Organisms 1 x 3-4 m, square ends, in long chains B. anthracis has poly-D-glutamic acid capsule Spores located in the center of nonmotile bacilli

Culture Colonies of B. anthracis round, a cut glass appearance in

transmitted light Hemolysis uncommon with B. anthracis Gelatin is liquefied, growth in gelatin stabs resembles an

inverted fir tree

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Bacillus SpeciesBacillus Species

B anthracis

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Bacillus SpeciesBacillus Species

BACILLUS ANTHRACIS IS A LARGE SPORE FORMING ROD WITH A PROMINANT POLY PEPTIDE CAPSULE

IT RAPIDLY BECOMES BLOOD BORN .THE SEPTICEMIC PHASE HAS HIGH MORTALITY.IT RARELY FORMS SPORE IN TISSUES. IT POSSESSES MULTIPLE VIRULENCE FACTORS.

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Bacillus Species, cont.Bacillus Species, cont.Growth Characteristics Simple sources of nitrogen and carbon for energy and

growth Spores are

– Resistant to environmental changes– Withstand dry heat and certain chemical disinfectants for

moderate periods– Persist for years in dry earth

Animal products contaminated with anthrax spores e.g. hides, bristles, hair, wool, bone can be sterilized only by autoclaving

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Bacillus anthracisBacillus anthracisPathogenesis Anthrax is primarily a disease of herbivores – goats, sheep,

cattle, horses– Rats relatively resistant to infection

Human infected incidentally by contact with infected animals or their products

In animals, portal of entry is the mouth and GIT Spores from contaminated soil ingested with spiny or

irritating vegetation In humans, infection acquired by the entry of spores thro

– Injured skin cutaneous anthrax– Rarely mucous membranes gastrointestinal anthrax– Inhalation of spores into the lung inhalation anthrax

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Bacillus anthracis, Bacillus anthracis, cont.cont. Spores germinate in the tissue at the site of entry Growth of vegetative organisms formation of a gelatinous edema and

congestion Spread via lymphatics to the bloodstream

– Multiply freely in the blood and tissues shortly before and after animal’s death

That does not produce capsule is not virulent Does not induce anthrax in test animals Capsule is antiphagocytic, gene on a plasmid Anthrax toxin (genes on plasmid) made up of 3 proteins

– Protective antigen (PA)– Edema factor (EF) and– Lethal factor (LF)

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Bacillus anthracis, Bacillus anthracis, cont.cont. PA binds to specific cell receptors, following proteolytic

activation it forms a membrane channel that mediates entry of EF and LF into the cell

EF is an adenylyl cyclase, with PA it forms a toxin known as edema toxin

LF plus PA form lethal toxin, a major virulence factor and cause of death in infected animals

Inhalation anthrax (Woolsorter’s disease) Spores from the dust of wool, hair or hides are inhaled Phagocytosed in the lungs, transported by the lymphatic

drainage to mediastinal lymph nodes, germination occurs toxin production, hemorrhagic mediastinitis, sepsis, fatal

In anthrax sepsis, # of organisms in blood >107/ml prior to death

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Bacillus anthracis, Bacillus anthracis, cont.cont.Pathology In susceptible animals, proliferate at the site of entry

– Capsule remain intact– Organisms surrounded by large amount of proteinaceous fluid containing few

leukocytes, rapidly disseminate and reach bloodstream In resistant animals, proliferate for a few hrs massive accumulation

of leukocytes Capsule gradually disintegrate and disappear, remain localizedClinical Findings In humans, 95% of cases are cutaneous anthrax

– 5% inhalational GI anthrax very rare, reported from Africa, Asia, USA, following

ingestion of meat from infected animals Bioterrorism events in the fall 2001

– 22 cases of anthrax; 11 inhalation, 11 cutaneous– 5 of inhalation anthrax died, the rest survived

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Bacillus anthracis, Bacillus anthracis, cont.cont.Cutaneous anthrax Occurs on exposed arms or hands, face and neck Pruritic papule develops 1-7 days after entry/spores

– Resembles an insect bite

Papule vesicle, coalesce necrotic ulcer– Lesions 1-3 cm diameter, central black eschar

Marked edema occurs Lymphangitis, lymphadenopathy, fever, malaise, headache After 7-10 days, eschar is fully developed It dries, loosens and separates, healing by granulation and

leaves a scar 20% of patients sepsis, meningitis and death

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Bacillus anthracis, Bacillus anthracis, cont.cont.Inhalation anthrax IP as long as 6 wks Marked hemorrhagic necrosis and edema of mediastinum

– Substernal pain may be prominent– Pronounced mediastinal widening visible on CXR

Hemorrhagic pleural effusion; cough secondary to effects on trachea

Sepsis occurs, hematogenous spread to GIT, meninges bowel ulceration, hemorrhagic meningitis

Fatality rate is high in the setting of known exposure Animals acquire anthrax through ingestion of spores, spread

from intestinal tract– Rare in humans, GI anthrax is extremely uncommon

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Bacillus anthracis, Bacillus anthracis, cont.cont.Diagnostic Lab TestsSpecimens Fluid or pus from a local lesion, blood, sputumMicroscopy Stained smears from dead animals large GPR In dried smears by immunofluorescence staining techniquesCulture On BA nonhemolytic gray to white colonies

– Comma-shaped outgrowth (Medusa head) may project In semisolid medium, always nonmotile B. cereus exhibit motility by swarming On bicarbonate-containing medium in 5-7% CO2

demonstration of capsule

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Bacillus anthracis, Bacillus anthracis, cont.cont. Lysis by a specific anthrax -bacteriophage helpful in

identification

Animal inoculation Virulent org. kill mice or guinea pigs on intraperitoneal inj.

Biochemical test Carbohydrate fermentation is not useful Gelatin liquefaction, index of proteolytic activity

Serology ELISA, not extensively studied Acute and convalescent sera in 4 wks apart should be tested

– Positive result is a 4-fold change– A single titer >1:32

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Bacillus anthracis, Bacillus anthracis, cont.cont.Resistance and Immunity Active immunity to anthrax induced in susceptible animals

by vaccination with – live attenuated bacilli, spore suspensions or PA from culture

filtrates Animals graze in known anthrax disease should be

immunized for anthrax annually Anthrax vaccine available for humans in US

– An aluminium hydroxide-precipitated preparation of PA from sterile filtrate of cultures of avirulent strain

– 2 inoculations yielded significant protection from inhalation anthrax in rhesus monkeys

In Russia, a live attenuated spore-based vaccine widely used in humans in large field trials

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Bacillus anthracis, Bacillus anthracis, cont.cont.Treatment Ciprofloxacin for treatment of anthrax Penicillin G, along with gentamicin or streptomycin

previously used to treat anthrax In the setting of potential exposure

– Prophylaxis with ciprofloxacin or doxycycline should be continued for 4 wks

– While 3 doses of vaccine are being given– Or for 8 wks if no vaccine is administered

B. cereus resistant to penicillin– Doxycycline, erythromycin or ciprofloxacin alternatives

to penicillin

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Bacillus anthracis, Bacillus anthracis, cont.cont.Epidemiology, Prevention and Control Soil is contaminated with anthrax spores from carcasses of dead

animals– Remain viable for decades– Can germinate in soil at pH 6.5 at proper temperature

Grazing animals infected thro injured mucous membranes serve to perpetuate chain of infection

Contact with infected animals or their hides, hair and bristles is the source of infection in humans

Control measures include: Disposal of animal carcasses by burning or by deep burial in lime pits Decontamination of animal products Protective clothing and gloves for handling potentially infected

materials Active immunization of domestic animals and high occupational risk

personnel with live attenuated vaccines

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Bacillus cereusBacillus cereus Produces toxins that cause disease that is more an

intoxication than a food-borne infection Food poisoning has two forms: emetic and diarrhoealEmetic type Commonly contaminates rice When large amount of rice are cooked and allowed to cool

slowly, spores germinate and vegetative cells produce toxin during log-growth phase or sporulation

Begins 1-5 hrs after ingestion of rice and occasionally pasta dishes

Nausea, vomiting, abdominal cramps, occasionally diarrhoea

Self-limiting, with recovery within 24 hrs

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Bacillus cereus, Bacillus cereus, cont.cont.Diarrhoeal type Associated with meat dishes and sauces Enterotoxin may be preformed in the food or produced in

the intestine IP 1-24 hrs Profuse diarrhoea with abdominal pain and cramps Fever and vomiting uncommon Presence of B. cereus in a patient’s stool not sufficient to

make a diagnosis– Bacteria may be present in normal stool specimens– A concentration of 105 bacteria or more/g of food is

diagnostic

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Bacillus cereus, Bacillus cereus, cont.cont. Important cause of eye infections, severe keratitis,

endophthalmitis and panophthalmitis Introduced into the eye by foreign bodies assoc. with trauma Also assoc with endocarditis, meningitis, osteomyelitis,

pneumonia– Presence of medical device or IVD use predisposes

Genes from B. thuringiensis coding for insecticidal compounds inserted into genetic material of some commerc. plants– Assoc with concern on environmental activitists about

genetically engineered plants and food products

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Bacillus cereus, cont.Bacillus cereus, cont.

B cereus