1. Spore-forming gram-positive bacilli
Bacillus (Aerobic)
B. antheracis, B.cereus, B. subtilis
Clostridum (Anaerobic)
C. tetani, C. botulinum, C. perfringens, C. difficile

2. Bacillus anthracis

3. Bacillus anthracis Disease
The anthrax bacillus, Bacillus anthracis, was the first bacterium shown to be the cause of a disease.
Disease
Anthrax (common in animal but rare in humans).
German bacteriologist Robert Koch confirmed bacterial origin of anthrax.
In Iran an anthrax outbreak killed more than 1 million sheep.

4. History (Recent years)
About 150,000 U.S. troops were vaccinated for anthrax in preparation for Persian Gulf War.
Letters containing anthrax spores were mailed to many places in the US such as NBC, New York Times, and Media in Miami.

5. Properties A large rod with square ends. Frequently in chains
µm in width x 3 - 5µm in length
Gram + rod
Facultative anaerobe
A large rod with square ends.
Frequently in chains

6. Properties
A unique anti-phagocytic capsule is composed of D-glutamate (Glutamyl-polypeptide capsule)
Non-motile (other members of the genus are motile.
Forms oval, centrally located endospores
Head medusa, rough, gray colonies
Inhalation Anthrax (2)

7. Bacillus cereus
Gram stain. The cells have characteristic squared ends. The spores are highly refractile to light and resistant to staining.

9. Robert Koch's original micrographs of the anthrax bacillus

14. Genetics 1 chromosome 5.2 million bp Ames strain sequenced 2 plasmids
px01
184 kbp
Exotoxin
pX02
95.3 kbp
Capsule

15. Anthrax From the Greek word anthrakos for coal Caused by spores
Primarily a disease of domesticated & 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
Anthrax zones
Soil rich in organic matter (pH < 6.0)

16. Transmission
May be spread by streams, insects, wild animals, birds, contaminated wastes
Animals infected by soilborn spores in food & water or bites from certain insects
Humans can be infected when in contact with flesh, bones, hair, & excrement
Risk of natural infection
Outbreaks occur in endemic areas after outbreaks in livestock

17. Transmission
Spores persist in soil for years. Infection from animal products (hides, bristles and wool), contact with sick animal.
Portals of entry: skin, mucous membranes, and respiratory tract.

18. Three forms of Anthrax Cutaneous anthrax Skin Most common
Spores enter to skin through small lesions
Inhalation anthrax
Spores are inhaled
The most lethal type of Anthrax.
Gastrointestinal (GI) anthrax
Spores are ingested
Oral-pharyngeal and abdominal

19. Pathogenesis The infectious dose of B. anthracis in humans:
Minimum infection dose of ~ 1,000-8,000 spores
LD50 of 8,000-10,000 spores for inhalation
Virulence depends on 2 factors:
Capsule (Invasiveness, not protective antigen)
Exotoxin

20. Capsule Protects against phagocytosis & lysis during vegetative state
Sticky, gelatinous polymer
pX02 plasmid
Made up of D-glutamic acid
Non-toxic on its own
Only encapsulated B. anthracis is virulent
Most important role during establishment of disease
Protects against phagocytosis & lysis during vegetative state

21. Exotoxin pX01 plasmid AB model: Binding & Activating 3 components:
Protective antigen (PA), edema factor (EF) , lethal factor (LF)
Make up 50% of proteins in the organism
EF: An adenylate cyclase
Components are individually non-toxic
LF+PA  lethal
EF+PA  edema
EF+LF  inactive
PA+LF+EF  edema & necrosis; lethal

22. Cutaneous Anthrax
95% of anthrax infections occur when the bacterium enters a cut or scratch on the skin due to handling of contaminated animal products or infected animals.
May also be spread by biting insects that have fed on infected hosts.
After the spore germinates in skin tissues, toxin production initially results in itchy bump that develops into a vesicle and then painless black ulcer.

23. Cutaneous Anthrax
A painless ulcer with black, necrotic eschar. Local edema. usually 1-3 cm in diameter.
Incubation period:
Usually an immediate response up to 1 day
Case fatality after 2 days of infection :
Untreated (20%)
With antimicrobial therapy (1%)

27. Cutaneous Anthrax (3)
CDC, Cutaneous Anthrax—Vesicle Development

28. Inhalation Anthrax Natural infection is extremely rare
Spores need to be less than 5 microns (millions of a meter) to reach the alveolus.
Macrophages lyse and destroy some of the spores.
Survived spores are transported to lymph nodes.

29. The two lungs are separated by mediastinum: Heart, trachea, esophagus, blood vessels.

30. Inhalation Anthrax (2)
Spores germinate and replicate in the lymph nodes.
Exotoxins: mediastinal widening and pleural effusions (accumulation of fluid in the pleural space).

31. Inhalation Anthrax (3)
Death usually results 2-3 days after the onset of symptoms.
Incubation period:
1–7 days
Possibly ranging up to 42 days (depending on how many spores were inhaled).
Case fatality after 2 days of infection:
Untreated (97%)
With antimicrobial therapy (75%)

33. Gastrointestinal Anthrax
GI anthrax may follow after the consumption of contaminated, poorly cooked meat.
There are 2 different forms of GI anthrax:
1) Oral-pharyngeal
2) Abdominal
Abdominal anthrax is more common than the oral-pharyngeal form.

34. GI Anthrax (2)
Oral-pharyngeal form - results from the deposition and germination of spores in the upper gastrointestinal tract.
Local lumphadenopathy (an infection of the lymph glands and lymph channels), edema, sepsis develop after an oral or esophageal ulcer.
Abdominal form - develops from the deposition and germination of spores in the lower gastrointestinal tract, which results in a primary intestinal lesion.
Symptoms such as abdominal pain and vomiting appear within a few days after ingestion.

35. This picture is 9 days after the onset of symptoms of oral-pharyngeal anthrax, an unusual manifestation of human infection with B. anthracis.

36. GI Infection (3) GI anthrax cases are uncommon. Incubation period:
1-7 days
Case fatality at 2 days of infection:
Untreated (25-60%)
With antimicrobial therapy (undefined) due to the rarity

37. The symptoms for Inhalation & Gastrointestinal anthrax
There are two phases of symptoms.
1) Early phase - Many symptoms can occur within 7 days of infection
2) 2nd phase - Will hit hard, and usually occurs within 2 or 3 days after the early phase.

38. Early Phase Symptoms Fever (temperature > 38 degrees celsius)
Chills or night sweats
Headache, cough, chest discomfort, sore throat
Joint stiffness, joint pain, muscle aches
Shortness of breath
Enlarged lymph nodes, nausea, loss of appetite, abdominal distress, vomiting, diarrhea
Meningitis

39. 2nd Phase Symptoms Breathing problems, pneumonia Shock
Swollen lymph glands
Profuse sweating
Cyanosis (skin turns blue)
Death

40. Lab. diagnosis Samples: Exudate, Blood, sputum.
Direct smear: Large rods in chains. Spores not seen in smears of exudate.
Culture and biological/biochemical tests (Sensitivity to penicillin (String of pearls test), Fermentation, gelatin hydrolysis, Motility)
No serological tests are useful

41. How is anthrax diagnosed?
Gram stain
Immunoflourescence staining
Culture of B. anthracis from the blood, skin lesions, vesicular fluid, or respiratory secretions
X-ray and Computed Tomography (CT) scan
Rapid detection methods
- PCR for detection of nucleic acid
- ELISA assay for antigen detection

42. Chest X-ray Useful for inhalation and GI anthrax
Chest X-rays is advised as an initial method of inhalation anthrax detections.
Find a widened mediastinum and pleural effusion.
Mediastinum is the area between the lungs.
A pleural effusion is an accumulation of fluid between the layers of the membrane that lines the lungs and chest cavity.
At day 1
At day 3

44. CT scan Useful for inhalation and GI anthrax
Chest CT (Right) shows the increase in the size of the pleural effusions (accumulation of fluid in the pleural space).

45. PCR Assay Used for the detection of anthrax toxin genes.
rpo B gene - used as a specific chromosomal marker for
RT-PCR detection.
Provides 100% sensitivity and specificity

46. Distinguishing inhalation Anthrax from cold or influenza
Anthrax, cold, and influenza patients have similar symptoms at early phase but Anthrax:
No runny nose
Breathing problems and more vomiting
High white blood cell counts and no increase in the number of lymphocytes
Inhalation anthrax has abnormality in X-ray or CT scan

47. Prevention Preventing soil contamination
Sterilizing dead animals and animal products
Protecting persons at risk of exposure with special clothes
Vaccination with cell-free vaccine (PA) for persons at high risk and animals

48. Who gets the vaccine? People working directly with it in the lab
People working with imported animal hides or furs in areas where standards are insufficient to prevent exposure
People handling potentially infected animal products in high-incidence areas
Military personnel deployed to areas with high risk for exposure

49. Treatment
Penicillin (Before 2001, 1st line of treatment was penicillin G but Stopped for fear of genetically engineered resistant strains)
Doxycycline
Ciprofloxacin (from fluoroquinolones)

50. Weaponization & Bacillus Anthracis:
Why is this Agent Considered to be the Department of Defense’s Number - One/Two Biological Threat?

51. Why are Biological Agents Attractive Weapons? (2)
Silent, Unnoticeable Attacks
Bombs & bullets are loud and there effects often dramatic and widely evident - not the case with BW
BW can be tasteless, odorless, colorless and unnoticeable
Allows for more facile attacks on large populations
People could be inflicted and not immediately realize it - time lag

52. Specific Benefits of Using Anthrax as a Biological Weapon
Short Incubation Period (Relative to Most Other BW)
Lag-time between attack and the first symptoms is only 1-6 days
Prediction of intended effect is much more facile to estimate
In contrast, bacterial agent brucellosis has an incubation of 5-60 days
UV Resistant
One of only two bacterial agents that is considered resistant to sunlight (the other being Coxiella)

53. How to Aerosolize?

54. Protection Against Anthrax
Pre/Post Exposure Antibiotic Treatment
Decontamination of Exposed Areas
Using liquid chlorine dioxide or some other disinfectant
Use of Protective Clothing & Equipment
Gas masks provide good protection against 1-5 m particles
Protective suits can be worn to easily eliminate cutaneous threat

55. Bacillus cereus
Motile
No capsule
Saprophyte

56. Laboratory differentiation of Bacillus anthracis & B. cereus
(phenethyl alcohol)

57. Bacillus cereus Disease Food poisoning
Rare infections: Meningitis, Osteomyelitis, …
Transmission
Spores on grains survive during steaming and rapid frying. Spore germinated when rice is reheated.
Portal of entry is the gastrointestinal tract.

58. Pathogenesis Clinical findings B. cereus produces 2 enterotoxins.
Emetic syndrome
A short incubation period (4 hours) with nausea and vomiting similar to staphylococcal food poisoning.
Diarrheal syndrome
Involves a long incubation period (18 hours) with diarrhea and resembles clostridial gastroenteritis.

59. Lab. diagnosis Treatment Prevention Not usually done
No antibiotic is given. Only symptomatic treatment
Prevention
Grains (specially rice) should not be reheated