Virulence factors of Bacillus anthracis

1. Bacillus anthracis Toxins
Dr Ravi Kant Agrawal, MVSc, PhD,
Senior Scientist (Veterinary Microbiology)
Food Microbiology Laboratory
Division of Livestock Products Technology
ICAR-Indian Veterinary Research Institute
Izatnagar 243122 (UP) India

2. Introduction
• Anthrax is a disease caused by Bacillus anthracis, a spore-forming, Gram
positive, aerobic, non-motile, rod-shaped bacterium.
• Bacillus anthracis produces a toxin composed of three distinct proteins.
• The toxin was first discovered by Harry Smith in 1954.
 In the bacteria there is one chromosome and 2 plasmids (pXO1 and pXO2)
• The lethality of the disease is caused by the bacterium's two principal
virulence factors:
 The tripartite protein toxin, called anthrax toxin are carried on a Large
plasmid (pX01).
 The polyglutamic acid capsule, which is anti-phagocytic are carried on a
second plasmid (pX02).
• AB model: B-Binding A- Activating
• Protective antigen (PA), edema factor (EF) & lethal factor (LF)
• Make up 50% of proteins in the organism
• Individually non-toxic
– PA+LF  lethal activity
– EF+PA  edema
– EF+LF  inactive
– PA+LF+EF  edema & necrosis; lethal

3. Bacillus anthracis
Bacillus anthracis exists in two forms:
1. Vegetative form
– 1 – 1.5 μm x 5 – 8 μm
– Square-ended rod
– Anaerobic environment of host
– Only multiply within host in the presence of amino
acids, nucleosides, and glucose
2. Inert spore form
– 1 μm
– Rod shaped
– Aerobic (free O2 present)
– Highly Resistant to extreme conditions (e.g.
temperature, weather, radiation, etc.)
– Endospore: ellipsoidal shaped centrally
– Spores have been known to last up to 40 yrs

4. Requirements for Pathogenesis
• Anthrax Toxin Receptor
• Capsule
• Toxins
Protective Antigen
Lethal Factor and/or
Oedema Factor

5. Anthrax Toxin Receptor (ATR)
• Most cell types can be intoxicated by anthrax toxin because
they express one or both of the cellular receptors of PA,
ANTXR1, and ANTXR2.
• Both are Type I membrane protein with an extracellular
domain that binds PA.
• The natural function of the anthrax receptors are to promote
cell adherence and spreading in angiogenic processes.
• Highly conserved between different species

6. Bacillus anthracis
• Chromosome  not involved in virulence
• pXO1  codes for PA, EF, and LF
• pXO2  codes for the capsule

7. pXO2/Capsule
•Glycocalyx
–Sticky, gelatinous polymer external to cell
wall
•pX02 plasmid
•96,231 bp (95kb), ~85 ORFs (113 genes)
•Contains capsule biosynthetic operon:
capBCA and and transcrtional regulator of
this operon, AcpA and AcpB.
•pXO2 codes for the homopolymeric γ-
linked poly-D-glutamic acid capsule
•Only encapsulated B. anthracis virulent
•Anti-phagocytic function
•Is non-toxic by itself
•Is important in the onset of the infection
•Most important role during establishment
of disease
–Protects against phagocytosis & lysis
during vegetative state
–Poorly understood
http://textbookofbacteriology.net/BS
RP.html

8. pXO1
• 181,654 nucleotides (182kb), ~143 ORFs (217 genes)
• Codes for the three toxin genes which are transcribed
simultaneously:
– pag A (for PA)
– cya (for EF)
– lef (for LF)
• Also codes for a germination operon (gerX) and
transcriptional regulatory proteins, from the atxA and PagR
genes.
• AtxA is master virulence regulator.
• AtxA causes ~10x increase in transcription of all three toxins.
• Anthrax toxin is a key virulence factor that is optimally
produced under host like conditions as
• C02> 5%, Temp 370
C.
• These conditions increase toxin gene expression through
master virulence regulator atxA.
• Co2 does not affect atxA levels and its MOA of enhanced toxin
expression is not clear.

9. Protective Antigen PA
• Is non-toxic without EF and LF
• Coded from pag gene
– A/T rich (69%)
– Cysteine-free
• 83 kDa (735-amino acid)
• Long, flat protein
• Function – to aid in the translocation of EF and LF
toxins in the cytosol

10. PA Structure
• Domain 1 (Green and Blue)
– residues 1 – 249, β-sandwich fold,
– 2 Ca2+
– forms cleavage site during
proteolytic activation
• Domain 2 (Magenta)
– Residues 250 – 487, β-barrel core
– Forms membrane inserted channel
/pore formation
• Domain 3 (Yellow)
– Residues 488 – 594
– 4-stranded mixed β-sheet, 4 small
helices
– Helps to oligomerize(?)
• Domain 4 (Purple)
– Residues 595 – 735, β-sandwich
– Contains receptor binding domain
M. Mourez et al. Trends Microbiol. 10:287-
293 (2002)

11. PA Mechanism
 PA83 binds to cell receptor
 Receptor bound PA is a substrate for cellular furin-like
proreases which cleaves PA83 between aa 164-167 (Arg-Lys-Lys-
Arg) to two fragments: PA20 (released) and PA63 (remains
attached to the receptor).
 PA63/receptor complexes join to form a HEPTAMER at reduced
pH.
 The heptamer complex can then bind to one LF or EF molecule
Koehler, T.M. Anthrax. Springer. 2002.

12. THE PA63 HEPTAMER
 LOSS OF PA20 LEADS TO
HEPTAMER FORMATION BY
PA63.
 HEPTAMER IS WATER SOLUBLE
AT NEUTRAL OR BASIC pH.
BUT
 HEPTAMER INSERTS INTO
MEMBRANE AT ACIDIC pH
FORMING CATION- SELECTIVE
CHANNELS IN BOTH ARTIFICIAL
LIPID BILAYERS AND CELLS.

13. PA Mechanism (cont.)
 The heptamer is internalized via RME.
 Acidification of the vesicle causes the heptamer to
form a pore in the membrane
 EF and LF are released through the pore into the
cytosol
http://www.biotechjournal.com/Pathways/anthrax.htm

14.  After PA binds to its receptor, host proteases cleave PA,
releasing a small fragment and retaining the 63-kDa fragment
(PA63) on the cell surface.
 The PA63 fragments and receptos co-oligomerizes (self-
associate) on the cell surface, forming a ring-shaped complex
of seven fragments- HEPTAMER (pore precursor or "prepore").
 This heptameric complex can then bind up to three molecules
of LF and/or EF.
 Both LF and/or EF factors recognize the same binding site of
PA63, so the binding is competitive.
 Formation of the complex stimulates endocytosis (RME) and
movement to an acidic compartment.
 In this environment, the heptameric complex forms a
transmembrane pore and releases LF and EF into the cell
cytosol.

15. General Mechanism
Koehler, T.M. Anthrax. Springer. 2002.

16. EF
• Coded from cya gene
– A/T rich (71%)
– Cysteine-free
• 89 kDa (767-amino acid)
• Mainly β strands
• A-B toxin
• Requires activation by calmodulin (CaM)
• N-terminal ~250 aa bind to PA63
• Remaining molecule possesses catalytic activity in
substrate and calmodulin-binding subdomains.
• Identical 7 PA binding residues as LF
• Impairs phagocytosis in macrophages
• Contributes to both cutaneous and systemic anthrax

17. EF Function
• EF receptors are present on most cells
• EF is a calmodulin dependent adenylate cyclase
– Therefore only functions in eukaryotic cells
• Catalyzes breakdown of ATP to cAMP
– ~200 fold increase
• Why Calmodulin?
– 1% total cellular protein
– Responsible for mediating Ca2+
signaling in cells

18. EF Mechanism
1. EF enters the cell by binding to PA
(called EdTx)
2. Inside, EF binds to calmodulin at the N-
terminal low Ca2+
affinity site in a very
unique way:
– One half grips lower lobe while the other
grasps the upper lobe and twists
– The method of binding forms a contact
region of 6,000 Å2
– an extremely large
binding region!
– The conformational change experienced
by both molecules inhibits Ca2+
from
binding and inhibits the reverse reaction
of cAMP to ATP
2. The conformational change in EF allows
it to act as an adenylate cyclase and bind to
ATP
3. cAMP is formed due to the binding of EF
to ATP
Liddington, R.C., A Molecular Full Nelson.
Nature. 415: 373-374, 2002.
The EF-calmodulin complex is composed
of many H- and ionic-bonds which form a
pocket in which ATP binds, Requires 1
metal ion in the cleft

19. EF
• What happens as a result of this activity?
– Causes edema by altering H2O and ion movement
– Inhibits immune response against Bacillus anthracis
– Inhibits activity of TF NF-ĸB in macrophages
– Strains with only EF and PA (LF -) are not likely to be lethal
http://www.biocarta.com/pathfiles/h_anthraxPathway.asp

20. • EF is a calmodulin-dependent adenylate cyclase that increases
the intracellular cyclic adenosine mono phosphate (cAMP)
levels, resulting in edema.
• EF is related to the adenylate cyclases produced by Bordetella
pertussis and Pseudomonas aeruginosa.

21. Oedema Factor Structure
• Active site in interface of
CA and CB
• Catalytic machinery is
present, but disordered
• CaM displaces helical
domain
• Switch B becomes
ordered
– binds ATP
– stabilizes EF catalytic residues
(inactive) (active)

22. More Fun with CaM binding
• Large binding surface stabilizes structural changes
• ATP locked into catalytic site by salt bridge
• Conformational changes to active site do not directly
involve catalytic residues
– become exposed to solvent in active state

23. Effects of EF Activation
• EF-CaM forms an irreversible complex
– CaM forced into extended conformation
• Adenylate cyclase becomes active
• Conversion of ATP  cAMP
• Increased [cAMP] perturbs immune effector cell functions
– Phagocytosis
– Chemotactic response
– Cytokine expression

24. LF
• Coded from lef gene
– A/T rich (70%)
– Cystenine-free
• 90.2-kDa (776-amino acid)
• A-B Toxin
• 3 domains:
– N-terminal ~250 aa competes
with EF to bind PA63
– 5 imperfect repeats,
each 19 aa long
– Contains a zinc protease
active site (HEXXH helix)
Pannifer, A.D., Wong, T.Y., et al, Crystal Structure of the Anthrax Lethal Factor. Nature. 414: 229-233, 2001.
Note: HEXXH (X is any aa) is characteristic of metalloproteases

25. LETHAL FACTOR
DOMAIN I: INVOLVED IN PA BINDING
DOMAIN II: RESEMBLES ADP
RIBOSYLATING TOXIN OF B. cereus,
AUGMENTS SUBSTRATE RECOGNITION
DOMAIN III: ALONGWITH DOMAIN 2 AND
4 HELPS IN HOLDING THE 16 RESIDUE
LONG N-TERMINAL TAIL OF MAPKK
BEFORE CLEAVAGE. POSSIBLY
INVOLVED IN MEMBRANE INSERTION.
DOMAIN IV: Zn CONTAINING CATALYTIC
SITE

26. LF Function
• LF only binds to macrophages
• LF is responsible for most of the toxicity
– Can induce lysis in about 90-120 minutes
• LF is a zinc dependent metalloprotease which cleaves
MAPKK
MAPKK: mitogen activated protein kinase kinase

27. LF Mechanism and Function
• LF is internalized via PA (called LeTx)
• Mechanism and Result of MAPKK cleavage:
– LF cleaves between 7-10 residues on the N-terminus of the
MAPKKs at a pair of proline residues (separtated by 0 – 4 aa)
• E.g. MAPKK1, 2, 3, 4, 6
– This cleavage serves to inactivate the MAPKK
– MAPKK cleavage leads to lysis of the macrophages, although
the mechanism is unclear
Duesbery, N.S., Vande Woude, G.F., Anthrax Toxins. Cellular and Molecular Life Sciences. 55: 1599-1609,
1999.
Note: MAPKK activation plays a role in cell division stress signals, (1-3)
production of cytokines and activation of macrophages

28. LF Mechanism and Func. (cont)
–Some MAPKK aid in the activation of
Trancription Factor -NF-ĸB
• This illustrates how EF and LF work
together to promote macrophage lysis
• At sublytic levels it stimulates
production of IL-1β and TNFα
– At lytic levels the mechanism is
unclear, but it is possible that in the
early stages they inhibit the immune
response to allow the bacteria to
proliferate, and at later stages it
stimulates them to cause lysis of cells
•Causes leakage in cell membrane
without ATP  increased Ca2+
conc. in
cell
•Strains with only LF and PA (EF -) are
likely to be lethal, although to a lesser
degree
http://www.biotechjournal.com/P
athways/anthrax.htm

29. LF
LF is zinc-dependent protease that is capable of cleaving
mitogen-activated protein (MAP) kinase, leading to cell death by
incompletely understood mechanisms.

30. Anthrax toxin is an A/B toxin
 Interestingly, each individual anthrax toxin protein is, in fact,
nontoxic.
 Toxic symptoms are not observed when these proteins are
injected individually into laboratory animals.
 However, the co-injection of PA and EF causes edema, and the
co-injection of PA and LF is lethal.
 The former combination is called edema toxin, and the latter
combination is called lethal toxin. Thus the manifestation of
physiological symptoms requires PA, in either case.

31. Mechanism of Infection
• Anthrax spores enter body
• Germinate and multiple in lymph nodes
• PA, EF, LF excreted from bacteria
• PA binds to either of the 02 receptors.
• PA nicked by furin-like protease
– 20-kDa segment off leaving 63-kDa peptide
– PA63 remain associated with the receptor and co-oligomerizes to form
a ring shaped Heptamer
• Heptamer binds to a maximum of 03 molecules of EF or LF on the flat upper
surface of the ring
• PA63 causes the associated receptors to enter a lipid raft and then be
internalized by clathrin dependent endocytosis
• Acidic environment of the endosome allows loops from the heptamer to
form a transmembrane β-barrel and enzymes to partially unfold in to a
molten globule state.
• Translocation of EF and LF through PA63 transmembrane pore is drivin by
proton motive force.
• LF or EF refolds after entering the cytosol.
• LF cleaves MAPKK 1 & 2
• EF stimulates cAMP

32. Anthrax Toxin Mechanisms (Figure 1)

33. Anthrax Toxin Mechanisms (Figure 2)

34. Review
• EF
– Increased production of
cAMP
– Causes swelling
– Inhibits immune response
• Allows for invasion!
• LF
– Cleaves MAPKKs
– Inhibits immune response
– Destroys macrophages
• Cells can’t fight off infection
 they Die!

35. Overall Mechanism Review
Duesbery, N.S., Vande Woude, G.F., Anthrax Toxins. Cellular and Molecular Life Sciences. 55: 1599-1609, 1999.

36. Mechanism of Action
of anthracis ToxinsB. Anthracis
EF
B
LF
B
B
LF
EF
LF
EF Endosome
Acidic
Environment
B
cAMP
MAPK
EDEMA
Increased expression
of pro-inflammatory
mediators
IMMUNE SUPPRESSION
WBCs do not divide in
the presence of
pathogens; overall
decrease in phagocytosis

37. Receptor
Adenylate
Cyclase
Nucleus
PKA Regulatory
Subunit
PKA
Catalytic
Subunit
Normal Signaling Pathway
Leads to a temporary
increase in pro-inflamatory
proteins and an overall
activation of the immune
system.

38. Mechanism of Action of Edema Toxin
Nucleus
PKA
Catalytic
Subunit
Cell Membrane – toxin has already entered through RME
EF
Over expression of cAMP
by edema factor A
Leads to an abnormal
increase in pro-inflamatory
proteins, increased
capillary permeability &
decrease in phagocytosis

39. Mechanism of Action of Lethal Toxin
Needed for cell division of WBCs
during an immune response
Needed for expression of TNF – α
Tumor necrosis factor which increases
chemotaxis and phagocytosis
LF
Protease
MAP
Kinase
Cell Membrane – toxin has already entered through RME
Degraded
MAP kinase
Protein
Due to LF protease activity MAP
kinase is not active and normal
cellular responses are lost

40. Clinical Features of B. Anthracis
 Anthrax can manifest itself in 3 forms: cutaneous,
gastrointestinal, and inhalational (pulmonary).
 The type contracted depends on the modes of transmission,
which include:
 Contact with infected tissues of dead animals (eg,
butchering, preparing contaminated meat)  Cutaneous
 Consumption of contaminated undercooked meat
Gastrointestinal
 Contact with contaminated hair, wool, or hides (during
processing) or contact with products made from them
Inhalational &/or Gastrointestinal
 Biological warfare mainly inhalational &/or
Gastrointestinal

41. Clinical Forms
Inhalational Anthrax:
 Endospores are introduced into the body and alveoli via inhalation.
(small size of spores permits this).
 Macrophages phagocytose and lyse endospores, and then travel to
regional lymph nodes.
 Spores germinate to become vegetative cells within macrophages; they
then leave macrophages and multiply within the lymphatic system (1-43
days).
 Bacteria enter the bloodstream and can lead to septic shock and
toxemia with system wide edema and necrosis; hematogenous spread
can lead to hemorrhagic meningitis.
 True pneumonia is rare w/ Inhalational anthrax but focal, hemorrhagic,
necrotizing pneumonic lesions may be observed.
 Major causes of death is compression of lungs and septic shock.

42. contd…
Inhalational Anthrax:
 Development of symptoms and pathogenesis occurs in 2
stages:
 Exhibition of cold and flu like symptoms malaise, fever, prominent
cough, nausea, vomiting, drenching sweats, dyspnea, chest pain, and
headaches
 Develops rapidly (hours) and is characterized by acute dyspnea,
subsequent cyanosis, pleural effusion, widened mediastinum,
pulmonary edema, hemorrhagic symptoms of stage 1 become severe
(>24hrs and leads to death)
 ID50 (infective dosage that will infect 50% of exposed) is 8,000-
50,000 spores.
 LD50 (Lethal dosage that will kill 50% of exposed) is 2,500-
55,000 spores.
 Untreated 80% mortality rate

43. Clinical Forms
Cutaneous Anthrax:
 Endospores enter body through the skin usually via preexisting
skin lesions, abrasions, and skin mucus membranes.
 Low-level germination and toxin production occurs at entrance
site leading to localized necrosis with eschar formation and
edema (massive in some cases).
 Eschar formation:
 Day 1 pruritic red papule appears at the initial site of infection
 Day 2 papule becomes round ulcer
 Day 3 plaque develops around edema site and central papule necrotizes,
ulcerates, and forms a normally painless black eschar
 After 1-2 weeks eschar falls off and leaves ulcer that can heal by week 3.

44. Contd…
Anthrax eschar, neckAnthrax eschar, neck
CDC Public Health Image Library,CDC Public Health Image Library,
number 1933number 1933
Edema around anthraxEdema around anthrax
lesionlesion
AB Christie, LiverpoolAB Christie, Liverpool
Anthrax eschar, neckAnthrax eschar, neck
CDC Public Health ImageCDC Public Health Image
Library, number 1934Library, number 1934

45. Contd…
Cutaneous Anthrax:
 Endospores are often phagocytosed by macrophages and carried
to local lymph nodes, which can result in painful lymphadenopathy
and lymphangitis.
 Hematogenous spread with resultant toxemia can occur, with
symptoms such as headache and fevers (up to 102 °F) although
such spread is not common with appropriate antibiotic therapy
 On very rare occasions system wide infection can occur and
results in death within days.
 ID50 & LD50 are unknown for cutaneous anthrax
 Untreated mortality rate is 20%
 With antibiotic treatment death is very rare.

46. Clinical Forms
Gastrointestinal Anthrax:
 Pathogenesis and statistics are unclear due to rarity of this
form.
 Vegetative cells from uncooked meat rather than spores
(germination unnecessary).
 In oral pharyngeal anthrax, bacteria enter via the oral or
pharyngeal mucosa mucosal ulcers can occur initially,
followed by local/regional lymphadenopathy and localized
edema.
 In abdominal anthrax, entrance occurs @ the ileum or
cecum Intestinal lesions, regional lymphadenopathy, and
edema of the bowel wall and ascites initially causes nausea,
vomiting, and malaise progressing into bloody diarrhea, acute
abdomen, or sepsis.
 Mortality may drop with antibiotic treatment (inconclusive).

47. Summary of Clinical Forms

48. Diagnosis
 Cutaneous anthrax can be distinguished by 2 key features:
painlessness of lesions and relatively large extent of associated
edema (presence of eschars).
 Because of rarity of gastrointestinal cases diagnosis focus is
limited. Onset of characteristic symptoms should signal patient to
consult a physician immediately for antibiotic treatment.
 Distinguishing features of inhalational anthrax: presence of
widened mediastinum and pleural effusions on chest radiograph or
CT (chest compound tomographic) scan with minimum evidence of
pneumonia.

49. Diagnosis
Inhalational Anthrax:
 Easier to diagnose when occurs in several suspicious cases than single case (or
at high risk situations postal employee)
 Chest x-ray showing a widened mediastinum, infiltrates, pleural effusion, etc.
= likely anthrax infection
 CT scan showing hyperdense hilar and mediastinal nodes (white arrow, middle diagram) +
extensive mediastinal edema = anthrax infection
 Molecular Biology tests only available at Laboratory Response Network (LRN)
labs but highly reliable
Pulmonary anthrax chest X-rayPulmonary anthrax chest X-ray
showing widened mediastinumshowing widened mediastinum
Dept of Radiological PathologyDept of Radiological Pathology
Armed Forces Institute ofArmed Forces Institute of
PathologyPathology

50. Brain of a person whoBrain of a person who
died from inhalationaldied from inhalational
anthraxanthrax
Normal BrainNormal Brain
The Human BrainThe Human Brain
This slide compares a normal brain to the brain of a person with
hemorrhagic meningitis who died from inhalational anthrax. Hemorrhagic
meningitis occurs in one half of cases as a result of the seeding of the
meninges.

51. Diagnosis
Distinguish Anthrax from Influenza-Like Illness (ILI):
 Early symptoms (fever, chills, myalgias, fatigue, malaise, and
nonproductive cough) are similar.
 ILI patients have rhinorrhea and this is uncommon in Anthrax
patients.
 Anthrax patients have shortness of breath, an uncommon trait
of ILI patients.
 Nausea and vomiting are more common in anthrax infection
 Abnormal chest radiographs and CT scans  Anthrax
infection

52. Post-exposure Prophylaxis: Treatment and Therapy
 Limited number of cases and data for treatment
 Antibiotics must be administered before critical toxin levels are
reached
 Early attempts (cutaneous) surgery, but fear of septicemia (early
20th
century)
 Pasteur, Jourbert, & Fortineau antibiotics (focus of modern
treatment now)
 Early therapy utilized Penicillin G procaine but has been shifted
to other antibiotics due to Penicillin-resistant strain appearance

53. Laboratory Diagnostic Tests
Specimens:-
Specimens to be
examined are fluid or
pus from a local lesion,
blood, CSF, and
sputum.
Gram Stain :-
 Gram stain shows
large gram-positive
rods.
Squared
ends
EndosporeEndospore
(resistant to(resistant to
staining)staining)

54. Bacillus anthracis is an aerobic, gram-positive, spore-forming, nonmotile
Bacillus species.
The nonflagellated vegetative cell is large (1-8 µm (microns) long, 1-1.5 µm
wide).
Spore size is approximately 1 µm.
Spores grow readily on all ordinary laboratory media at 37°C.

55. Laboratory Diagnostic TestsDirect
Examination :
 Stained smears from the local lesion or of blood from dead
animals often show chains of large gram-positive rods.
 Carbohydrate fermentation is not useful.
 Anthrax can be identified in dried smears by
immunofluorescence staining techniques.
immunofluorescence staining of sporation

56. Laboratory Diagnostic Tests
Culture :
 Nutrient broth: Non-motile
 On blood agar plates, the
organisms produce non-hemolytic,
grey to white Rough, colonies
 On Mixed Flora a rough texture
and a ground-glass/cut-glass
appearance.
• Comma-shaped, irregular, curving
outgrowths from the margin of the
colonies - Medusa head colonies
 Selective Medium: PLET agar
(Polymyxin-Lysozyme-EDTA-
Thallous acetate)

57. Observation of colony
Enhance production of poly -D-glutamyl capsule
Increased CO2 tension to 5%Increased CO2 tension to 5%
Mucoid colony type (thick capsule)Mucoid colony type (thick capsule)

58. Positive encapsulation test for Bacillus anthracis
This slide shows blood agar and bicarbonate agar plate cultures of Bacillus
anthracis resulting in a positive encapsulation test for B. anthracis.
The rough colonies are on blood agar and smooth colonies on bicarbonate
agar.

59. Causative Organism
• Growth in gelatine
stabs resembles an
inverted fir tree.

60. Laboratory Diagnostic Tests

61. Laboratory Diagnostic Tests
Lab Characters
 Virulent anthrax cultures kill
mice upon intra-peritoneal
injection.
 Demonstration of capsule
requires growth on
bicarbonate-containing
medium in 5–7% CO2.
 Lysis by a specific anthrax
-bacteriophage may be helpful
in identifying the organism.

62. Treatment and Therapy cont…
 Fluoroquinone class antibiotics are now used Ciprofloxacin
in conjunction w/ Doxycycline as first line
Usual dosages:
 Cutaneous: adults & pregnant women 500mg Ciprofloxacin
and 100mg Doxycycline twice daily; children varies, based on
age and weight (All for at least 60 days).
 Inhalational & Gastrointestinal via IV: adults and pregnant
women 400mg Ciprofloxacin OR 100mg Doxycycline every
12hrs AND 2 additional antimicrobials (rifampin, vancomycin,
penicilin, ampicillin, chloramphenicol, imipenem, clindamycin, &
clarithromycin); children Ciprofloxacin OR Doxycycline varies,
based on age & weight AND one or two of the previously
mentioned antimicrobials.
 Therapy switched to oral treatment when clinically
appropriate and lasts for at least 60 days.

63. CategoryCategory Initial IV TherapyInitial IV Therapy DurationDuration
AdultsAdults Ciprofloxacin 400 mg everyCiprofloxacin 400 mg every
12 hr OR12 hr OR
Doxycycline 100mg everyDoxycycline 100mg every
12 hr12 hr
ANDAND
Additional 1 or 2 antibioticsAdditional 1 or 2 antibiotics
When clinically appropriateWhen clinically appropriate
switch to oral therapy:switch to oral therapy:
Ciprofloxacin 500mg 2xCiprofloxacin 500mg 2x
daily ORdaily OR
Doxycycline 100 mg 2xDoxycycline 100 mg 2x
dailydaily
Continue oral or IV therapyContinue oral or IV therapy
for 60 daysfor 60 days
Children*Children* Ciprofloxacin 10-15 mg/kgCiprofloxacin 10-15 mg/kg
ever 12 hr ORever 12 hr OR
Doxycycline:Doxycycline:
>8y and > 45kg: 100mg>8y and > 45kg: 100mg
every 12 hrevery 12 hr
>8y and>8y and << 45kg: 2.245kg: 2.2
mg/kg every 12 hrmg/kg every 12 hr
<8y 2.2mg/kg every 12<8y 2.2mg/kg every 12
hrhr
AND 1 or 2 additionalAND 1 or 2 additional
antibioticsantibiotics
Switch to oral whenSwitch to oral when
clinically appropriateclinically appropriate
Ciprofloxacin 10-15 mg/kgCiprofloxacin 10-15 mg/kg
every 12 hr ORevery 12 hr OR
Doxycycline:Doxycycline:
>8y and > 45kg: 100mg>8y and > 45kg: 100mg
2x daily2x daily
>8y and>8y and << 45kg: 2.245kg: 2.2
mg/kg 2x dailymg/kg 2x daily
<8y 2.2mg/kg 2x daily<8y 2.2mg/kg 2x daily
Continue oral or IV for 60Continue oral or IV for 60
daysdays
Pregnant Women*Pregnant Women* Same for non-pregnantSame for non-pregnant
adultsadults
Same for non-pregnantSame for non-pregnant
adultsadults
*Although ciprofloxacin and certain tetracyclines are not recommended for children and/or pregnant women, in life
threatening circumstances their use would be warranted. Adapted from CDC.

64. Anthrax Vaccines
 1881 Pasteur develops first live attenuated
veterinary vaccine for livestock
 1939 Improved live veterinary vaccine
 1954 First cell-free human vaccine
 1970 Improved cell-free vaccine licensed

65. Anthrax Vaccines
Preparation:
Immunization to prevent anthrax is
based on the classic experiments of
Louis Pasteur.
In 1881 he proved that cultures grown
in broth at 42–52 °C for several months
lost much of their virulence
be injected live into sheep and cattle
without causing disease; subsequently,
such animals proved to be immune.
Louis Pasteur

66. Anthrax Vaccines
 Four countries produce
vaccines for anthrax.
 Russia and China use
attenuated spore-based
vaccine administered by
scarification.
 The US and Great Britain
use a bacteria-free
filtrate of cultures
adsorbed to aluminum
hydroxide

67. Anthrax Vaccines
Pre-exposure Vaccination
 The current US FDA approved vaccine contains cell-free
filtrates of a toxigenic non-encapsulated non-virulent strain of
B. anthracis.
 The vaccine is available only to the US Department of Defense
and to persons at risk for repeated exposure to B anthracis.

68. Pre-exposure Prophylaxis: Anthrax Vaccine
 1881: first developed by Louis Pasteur
 1937: vaccine modified to use attenuated non pathogenic strain of B.
anthracis w/ dormant spores (other than former Soviet Union and associate
countries, this vaccine is preferred over live attenuated vaccine)
 U.S. currently uses AVA (Anthrax Vaccine Adsorbed) k/a BioThrax™ licensed
in 1970 and produced by Bioport Corp.
AVA: made from cell-free infiltrates of microaerophilic cultures of an
avirulent, nonencapsulated strain of B anthracis concentrated with PA
(protective antigen) final no live or dead bacteria are in final product
Final product contains: 1.2 mg/mL aluminum, added as aluminum hydroxide in
0.85% sodium chloride (Al = adjuvant that increase immune response); & 25
mcg/mL benzethonium chloride and 100 mcg/mL formaldehyde, added as
preservative
How efficacious? : several animal studies & 1 controlled human trial
induced immune response in 83% of adults vaccinated with one dose and 91% for
2 or more doses. ** Correlation between antibody titer and protection against
infection not yet defined.
Efficacy duration unknown, but animal tests suggest 1-2 years after 2 doses.
Vaccine not licensed for children due to lack of any pediatric testing, but
likely to be safe based on results with other inactive vaccines.
1998: Department of Defense makes vaccination for anthrax mandatory for
all troops (AVIP) over 400 quit or court marshaled due to fear of adverse side
effects.

69. Anthrax Vaccine cont…
Dosage:
 In U.S.: 6 doses of 0.5 mL at weeks 0,
2, & 4 AND months 6, 12, & 18, as well
as annual boosters
 In U.K.: 6 doses of 0.5 mL at weeks 0,
3, 6, & 32 with annual boosters.
Post-exposure vaccination:
 Vaccine can be used with antibiotics
after inhalation exposure
 3-dose regime @ weeks 0, 2,& 4 in
combination with at least 30-days of
antimicrobial therapy (w/out vaccine
60-day antimicrobial therapy is
needed)
 Vaccine not licensed for post-exposure
therapy.
 Therefore, it is administered as an
investigational new drug (IND) under
FDA regulations.

70. Anthrax Vaccine cont…
 Adverse effects:
 USAMRIID data: inflammatory reaction greater than 5 cm
in diameter around the site of injection found in about 2.4 -
3.9% of cases
 1990-200: Vaccine Adverse Event reporting System (VAERS)
receives 1,544 reports of adverse events after
vaccination most frequent included: injection site
hypersensitivity (334), injection site edema (283), injection
site pain (247), headache (239), arthralgia (232), asthenia
(215), and pruritus (212).

71. Anthrax Vaccine cont…
 Possible future Improvements on vaccine:
Cloning the PA gene into organisms of low pathogenicity, such as B.
subtilis, and creating recombinant vaccines
Creating mutant strains of vaccines that utilize non-human aromatic
compounds for virulence may establish better immunicity (minimizes
self-cell recognition problems) w/out risk of deadly infection
Experimenting with purified PA preparations with different
combinations of adjuvant molecules (similar to AVA approach)
1) PA with monophosphoryl lipid A (MPL) no cold storage chain required
and possibly more efficacious in conferring immunity
2) expression of the cereolysin AB gene in B. anthracis gives hemolytic
properties to the bacteria; conferred immunity against both H-7 strain wild
type and H-7 AB recombinant when immunized by recombinant STI-1 strain
containing the cereolysine AB gene
3) AVA may be more effective 100% immunity in 2-dose immunized rhesus
monkeys when compared to 100% mortality for non-immunized monkeys

72. Epidemiology
 Soil is contaminated with anthrax
spores from the carcasses of dead
animals.
 These spores remain viable for
decades. Perhaps spores can
germinate in soil at pH 6.5 at proper
temperature.
 Grazing animals infected through
injured mucous membranes serve to
perpetuate the chain of infection.

73. Prevention & Control
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 with live attenuated
vaccines.
Persons with high occupational risk should be immunized.

74. Thanks
References: All the presentations available online on the subject are
duly acknowledged.
Disclaimer: The author bear no responsibility with regard to the
source and authenticity of the content.