5. TROPIS 5 2012 (Bacillus Anthracis)

Bacillus anthracis Bacillus anthracis

Prof. Dr. Mochammad Hatta, Prof. Dr. Mochammad Hatta, PhD, PhD, SpMKSpMK (K) (K)Bagian MikrobiologiBagian MikrobiologiFakultas KedokteranFakultas Kedokteran

Universitas HasanudddinUniversitas HasanudddinMakassar Makassar

mochammadhatta@2013 mochammadhatta@2013

Anthrax is the preferred biological Anthrax is the preferred biological warfare agentwarfare agent

It is highly lethalIt is highly lethal. . • 100 million lethal doses per gram of anthrax material (100,000 • 100 million lethal doses per gram of anthrax material (100,000

times deadlier than the deadliest chemical warfare agent).times deadlier than the deadliest chemical warfare agent). • • Silent, invisible killer. Silent, invisible killer. • • Inhalational anthrax is virtually always fatal. Inhalational anthrax is virtually always fatal.

There are low barriers to production. There are low barriers to production. • Low cost of producing the anthrax material. • Low cost of producing the anthrax material. • Not high-technology. Knowledge is widely available. • Not high-technology. Knowledge is widely available. • Easy to produce in large quantities. • Easy to produce in large quantities.

It is easy to weaponizeIt is easy to weaponize. . • It is extremely stable. It can be stored almost indefinitely as a dry • It is extremely stable. It can be stored almost indefinitely as a dry powder. powder. • It can be loaded, in a freeze-dried condition, in munitions or • It can be loaded, in a freeze-dried condition, in munitions or disseminated as an aerosol with crude sprayers. disseminated as an aerosol with crude sprayers.

Currently, we have a limited detection capability. Currently, we have a limited detection capability. mochammadhatta@2013 mochammadhatta@2013

What is Anthrax?What is Anthrax?

Anthrax is a naturally occurring disease of Anthrax is a naturally occurring disease of plant eating animals (goats, sheep, cattle, plant eating animals (goats, sheep, cattle, wine, etc.) caused by the bacterium wine, etc.) caused by the bacterium Bacillus anthracis. Bacillus anthracis.


Sifat dan morfologiSifat dan morfologiVVery large, Gram-positive, sporeforming rod, 1 - ery large, Gram-positive, sporeforming rod, 1 - 1.2µm in width x 3 - 5µm in length. The 1.2µm in width x 3 - 5µm in length. The bacterium can be cultivated in ordinary nutrient bacterium can be cultivated in ordinary nutrient medium under aerobic or anaerobic conditions. medium under aerobic or anaerobic conditions. Genotypically and phenotypically it is very Genotypically and phenotypically it is very similar to similar to Bacillus cereus,Bacillus cereus, which is found in soil which is found in soil habitats around the world, and to habitats around the world, and to Bacillus Bacillus thuringiensis,thuringiensis, the pathogen for larvae of the pathogen for larvae of Lepidoptera.Lepidoptera. The three species have the same The three species have the same cellular size and morphology and form oval cellular size and morphology and form oval spores located centrally in a nonswollen spores located centrally in a nonswollen sporangium. sporangium.


VVirulence of irulence of Bacillus anthracisBacillus anthracis is is attributable to three bacterial attributable to three bacterial

components:components:

Capsular material composed of poly-D-Capsular material composed of poly-D-glutamate polypeptide glutamate polypeptide EF EF ((edema factoredema factor)) component of exotoxin component of exotoxinLF LF ((lethal factorlethal factor) ) component of exotoxin component of exotoxin


Robert Koch's Robert Koch's originaloriginal photomicrographs of photomicrographs of Bacillus anthracis,Bacillus anthracis, the the agent of anthrax. Look for large cells with square ends and agent of anthrax. Look for large cells with square ends and centrally-locatedcentrally-located ellipsoid spores when ellipsoid spores when iidentifyingdentifying Bacillus Bacillus anthracisanthracis


Bacillus anthracis. Bacillus anthracis. Gram stain. 1500X. The cells have Gram stain. 1500X. The cells have characteristic squared ends. The endospores are ellipsoidal characteristic squared ends. The endospores are ellipsoidal shaped and located centrally in the sporangium. The spores shaped and located centrally in the sporangium. The spores are highly refractile to light and resistant to staining.are highly refractile to light and resistant to staining.


Bacillus anthracis


Bacillus thuringiensis.Bacillus thuringiensis. Phase Photomicrograph of Phase Photomicrograph of vegetative cells, intracellular spores (light) and vegetative cells, intracellular spores (light) and parasporal crystals (dark). 1000Xparasporal crystals (dark). 1000X


Bacillus cereus. Bacillus cereus. Gram stain. 450X. Bacilli are large bacteria, so that Gram stain. 450X. Bacilli are large bacteria, so that they are readily observed with the microscope's "high dry they are readily observed with the microscope's "high dry objective" objective" ,,but can't detect anything about their spores.This could be but can't detect anything about their spores.This could be a a Lactobacillus.Lactobacillus.


Differential Characteristics of Differential Characteristics of B. anthracis B. cereus B. anthracis B. cereus

and B. thuringiensisand B. thuringiensis CharacteristicCharacteristic B. AnthracisB. Anthracis B. cereus B. cereus andand

B.thuringiensisB.thuringiensisgrowth requirementgrowth requirementfor thiaminfor thiamin ++ --

hemolysis on sheephemolysis on sheepblood agarblood agar -- ++

glutamyl-polypeptide capsuleglutamyl-polypeptide capsule ++ --

lysis by gamma phagelysis by gamma phage ++ --

MotilityMotility -- ++

growth on chloralhydrate agargrowth on chloralhydrate agar -- ++

string-of-pearls teststring-of-pearls test ++ --mochammadhatta@2013 mochammadhatta@2013

Colonies of Colonies of Bacillus cereusBacillus cereus on the left; colonies of on the left; colonies of Bacillus Bacillus anthracisanthracis on the right. on the right. B. cereusB. cereus colonies are larger, more mucoid, colonies are larger, more mucoid, and this strain exhibits a slight zone of hemolysis on blood agar.and this strain exhibits a slight zone of hemolysis on blood agar.


Lysis of Lysis of Bacillus anthracisBacillus anthracis by the lytic phage gamma. The plaque (clear area) by the lytic phage gamma. The plaque (clear area) in the region of confluent growth is where the gamma phage was applied. in the region of confluent growth is where the gamma phage was applied. The plaque results from the phage's abliity to lyse the bacterial cells. Since The plaque results from the phage's abliity to lyse the bacterial cells. Since the gamma phage is specific for the gamma phage is specific for B. anthracis,B. anthracis, and will not lyse and will not lyse B. B. thuringiensisthuringiensis or or B. cereus,B. cereus, we know that this is we know that this is Bacillus anthracisBacillus anthracis


Mucoid colonies of Mucoid colonies of Bacillus anthracis.Bacillus anthracis. This culture was This culture was probaby incubated at an increased CO2 tension (5% CO2) probaby incubated at an increased CO2 tension (5% CO2) which greatly enhances production of the poly-D-glutamyl which greatly enhances production of the poly-D-glutamyl capsule and accounts for the mucoid colony type.capsule and accounts for the mucoid colony type.


Two microscopic techniques to demonstrate the presence of Two microscopic techniques to demonstrate the presence of the poly-D-glutamyl capsule of the poly-D-glutamyl capsule of Bacillus anthracis.Bacillus anthracis. Left. India Left. India ink capsule outline 1000X. Right a fluorescent-labeled antibody ink capsule outline 1000X. Right a fluorescent-labeled antibody is reacted specifically with the capsular material which renders is reacted specifically with the capsular material which renders the capsule fluorescent - FA stain 1000X.the capsule fluorescent - FA stain 1000X.


McFadyean's reaction showing short chains of McFadyean's reaction showing short chains of Bacillus Bacillus anthracisanthracis cells lying among amorphous, disintegrated capsular cells lying among amorphous, disintegrated capsular material. White blood cells can also be seenmaterial. White blood cells can also be seen


The infectious dose of The infectious dose of B. anthracisB. anthracis and the lethal and the lethal dose of toxin varies greatly within animal species. dose of toxin varies greatly within animal species.

Animal Infect dose Toxic dose Bacteria per ml

model causing death blood at time death

Mouse 5 cells 1000 units/kg 107

Monkey 3000 cells 2500 unit/kg 107

Rat 106 cells 15 units/kg 105


Humans can contract anthrax in three ways: Humans can contract anthrax in three ways:

Through cuts or breaks in the skin resulting from Through cuts or breaks in the skin resulting from contact with an infected animal (cutaneous anthrax), contact with an infected animal (cutaneous anthrax), resulting in local and possibly systemic (bloodstream) resulting in local and possibly systemic (bloodstream) infection. infection. From breathing anthrax spores (termed "woolsorters" From breathing anthrax spores (termed "woolsorters" disease) resulting in an infection of the lungs disease) resulting in an infection of the lungs (inhalational anthrax). (inhalational anthrax). From eating infected meat, resulting in gastrointestinal From eating infected meat, resulting in gastrointestinal infection (gastrointestinal anthrax). Gastrointestinal infection (gastrointestinal anthrax). Gastrointestinal anthrax is generally not considered a threat to U.S. anthrax is generally not considered a threat to U.S. forces. forces.


What are the symptoms?What are the symptoms?

1 to 6 day incubation 1 to 6 day incubation periodperiodcutaneous anthrax, itching will occur at the cutaneous anthrax, itching will occur at the site of exposure followed by the formation site of exposure followed by the formation of a lesion. of a lesion. for inhalational anthrax are generally non-for inhalational anthrax are generally non-specific: low grade fever, a dry hacking specific: low grade fever, a dry hacking cough, and weakness cough, and weakness respiratory distress occurs with shock and respiratory distress occurs with shock and death following shortly thereafter death following shortly thereafter


Treatment of AnthraxTreatment of Anthrax

Antibiotics should be given to Antibiotics should be given to unvaccinated individuals exposed to unvaccinated individuals exposed to inhalation anthrax. Penicillin, tetracyclines inhalation anthrax. Penicillin, tetracyclines and fluoroquinolones are effective if and fluoroquinolones are effective if administered before the onset of lymphatic administered before the onset of lymphatic spread or septicemia, estimated to be spread or septicemia, estimated to be about 24 hours. about 24 hours.


PencegahanPencegahan

VaccinesVaccines composed of killed bacilli and/or composed of killed bacilli and/or capsular antigens produce no significant capsular antigens produce no significant immunity. A nonencapsulated toxigenic immunity. A nonencapsulated toxigenic strain has been used effectively in strain has been used effectively in livestock. The livestock. The Sterne StrainSterne Strain of of Bacillus Bacillus anthracisanthracis produces sublethal amounts of produces sublethal amounts of the toxin that induces formation of the toxin that induces formation of protective antibody protective antibody


Penanganan AnthraxPenanganan AnthraxSpores can only be destroyed by steam sterilization or Spores can only be destroyed by steam sterilization or burning. Chemical disinfection of buildings is burning. Chemical disinfection of buildings is problematic. The U.S. Navy Manual on Operational problematic. The U.S. Navy Manual on Operational Medicine and Fleet Support, entitled Biological Warfare Medicine and Fleet Support, entitled Biological Warfare Defense Information Sheet states "Disinfection of Defense Information Sheet states "Disinfection of contaminated articles may be accomplished using a contaminated articles may be accomplished using a 0.05% hypochlorite solution (1 tbsp. bleach per gallon of 0.05% hypochlorite solution (1 tbsp. bleach per gallon of water). Spore destruction requires steam sterilization." water). Spore destruction requires steam sterilization." Quarantine of affected individuals is not recommended Quarantine of affected individuals is not recommended A segment of the U.S. military population has been A segment of the U.S. military population has been vaccinated against anthrax. Anthrax vaccine consists of vaccinated against anthrax. Anthrax vaccine consists of a series of six doses with yearly boosters a series of six doses with yearly boosters


Documents

5. TROPIS 5 2012 (Bacillus Anthracis)