Bacillus and Biological warfare

Dr. Samah Binte Latif

M - Phil (part-1), Microbiology
Dhaka Medical College
 Introduction
• There are five medically important genera of gram-
positive rods: Bacillus, Clostridium, Corynebacterium,
Listeria, and Gardenella.

• Bacillus and Clostridium form spores, whereas

Corynebacterium, Listeria, and Gardenella do not.
Spores are never found in the animal body during life
but are formed in culture or in the soil.
• Most of the members of the Bacillus species are
saprophytic organisms prevalent in soil, water, air
and on vegetation (e g - B. subtilis).

• The genus Bacillus includes large aerobic, Gram-

positive rods arranged in chains. The members of this
genus are closely related but differ both
phenotypically and in terms of pathogenesis.
 Cntd….
• Pathogenic species possess virulence plasmids.

• Some are insect pathogens, such as B. Thuringiensis.

It is closely related to B. cereus and may occasionally
produce food poisoning. It is also used as larvicidal
agent for mosquito control.
There are about 70 or more species of the Bacillus
found in soil and water. Among them -
Pathogenic:
1.Bacillus anthracis
2.Bacillus cereus
Non pathogenic:
B.subtilis
B.lichenisformis
B.pumilis
B.Circulans
B.mycoides
 History of Bacillus anthracis
• B.anthracis is of considerable historical interest.
• It was the first pathogenic bacterium to be seen under the
microscope ( Pollender,1849).
• It was the first communicable disease to be transmitted
experimentally by inoculation of infected blood
( Davaine,1850).
• It was the first bacterium to be isolated in pure culture and
shown to possess spores ( Robert Koch,1876).
• Also it was the first bacterium employed for preparation of
live attenuated vaccine (Louis Pasteur,1888).
 Properties of Bacillus anthracis
1.Bacillus anthracis is a large gram-positive rod
( measuring 1ꭙ 3-4 µm) with square ends, frequently
found in chains. Spores are located in the center of the
bacilli.

2.It is non motile, whereas other members of the genus

are motile and non acid -fast.
3.Its anti-phagocytic capsule is polypeptide which is
composed of D-glutamate.

4. Anthrax toxin is encoded on one plasmid ( pX01) ,

and the polyglutamate capsule is encoded on a
different plasmid ( pX02).
 Virulence factors
Pathogenesis of anthrax is due to two important
virulence factors—anthrax toxin and capsule. Anthrax
Toxin consisting of three proteins.

1. Edema factor: It is the active part; acts as adenylyl

cyclase and increases host cell cAMP (cyclic
adenosine monophosphate). It is responsible for
edema and other manifestations seen in anthrax.
2. Protective factor:
It is the binding fragment that binds to the host cell
receptors and facilitates the entry of other fragments
into the host cells
3. Lethal factor: It causes cell death; acts by cleaving
host cell MAPK (mitogen-activated protein kinases).
• These fragments are not toxic individually, but in
combination, they produce local edema and
generalized shock. Toxin synthesis is controlled by a
plasmid (pX01). Loss of plasmid makes the strain
avirulent.
Capsule :
B. anthracis has a polypeptide capsule, made up of
polyglutamate.™Capsule is plasmid (pX02) encoded.It
inhibits complement mediated phagocytosis.
 Transmission to animals

Anthrax is primarily a zoonotic disease. Herbivorous

animals such as cattle, sheep and less often horses and
pigs are affected more commonly than the carnivorous
animals. ™ Infection occurs in susceptible animals by
ingestion of the spores present in the soil.
Direct spread from animal to animal is rare ™ Anthrax
in animals presents as a fatal septicemia; however,
localized cutaneous lesions may be produced rarely ™
infected animals discharge large number of bacilli from
the mouth, nose and rectum. These bacilli sporulate in
soil and remain as the source of infection for man.
 Transmission to humans
Spores of the organism persist in soil for years.
Transmission occurs in 3 routes:
1.Cutaneous:
Humans are most often infected cutaneously at the time
of trauma to the skin, which allows the spores on animal
products, such as hides, bristles, and wool, to enter.
2.Inhalation:
Spores can also be inhaled into the respiratory tract.
Pulmonary (inhalation) anthrax occurs when spores are
inhaled into the lungs.
3.Ingestion:
Gastrointestinal anthrax occurs when contaminated meat is
ingested.
• Inhalation anthrax is not communicable from person to
person, despite the severity of the infection.

• After being inhaled into the lung, the organism moves

rapidly to the mediastinal lymph nodes, where it causes
hemorrhagic mediastinitis. Because it leaves the lung so
rapidly, it is not transmitted by the respiratory route to
others.
• A 4th category of the disease, Injection anthrax, has
caused outbreak among persons who inject heroin
that has been contaminated with anthrax spores.
• The spores germinate in the tissue at the site of entry
and growth of the vegetative organisms results in
formation of a gelatinous edema and congestion.
• Bacilli spread via lymphatics to the bloodstream and
they multiply freely in the blood and tissues shortly
before and after the animal‫י‬s death.
 Pathogenesis of Bacillus anthracis
Pathogenesis is based primarily on the production of
two exotoxins, collectively known as anthrax toxin. The
two exotoxins, edema factor and lethal factor, each
consist of two proteins in an A–B subunit configuration.
The B, or binding, subunit in each of the two exotoxins
is protective antigen. The A, or active, subunit has
enzymatic activity.
 Cntd….
The protective antigen binds to a receptor on
the host cell membrane.

After proteolytic activation, it forms a pore on

the host cell membrane

Through which Edema factor and Lethal factor

enter into cell.
• Edema factor is an adenylate cyclase with PA, it
forms a toxin known as edema toxin that causes an
increase in the intracellular concentration of cyclic
adenosine monophosphate (AMP). This causes an
outpouring of fluid from the cell into the extracellular
space, which manifests as edema.
LF plus PA form lethal toxin, which is a major virulence
factor and cause of death in infected animals and
humans.

Lethal factor is a protease that cleaves the phosphokinase

that activates the mitogen-activated protein kinase
(MAPK) signal transduction pathway. This pathway
controls the growth of human cells, and cleavage of the
phosphokinase inhibits cell growth, leading to apoptosis,
cell death and the necrotic skin lesion( black eschar).
 In cutaneous anthrax
Cutaneous anthrax generally occurs on exposed
surfaces of the arms or hands followed in frequency by
the face and neck. A pruritic papule develops 1–7 days
after entry of the organisms or spores through a
scratch. Initially, it resembles an insect bite.
The papule rapidly changes into a vesicle or small ring
of vesicles that coalesce, and a necrotic ulcer develops.
The lesions typically are 1–3 cm in diameter and have a
characteristic central black eschar. Marked edema
occurs. Lymphangitis, lymphadenopathy, and systemic
signs and symptoms of fever, malaise, and headache
may occur.
After 7–10 days, the eschar is fully developed.
Eventually, it dries, loosens and separates; healing is by
granulation and leaves a scar. It may take many weeks
for the lesion to heal and the edema to subside.
Antibiotic therapy does not appear to change the
natural progression of the disease but prevents
dissemination. In as many as 20% of patients,
cutaneous anthrax can lead to sepsis, the consequences
of systemic infection including meningitis and death.
 Pathogenesis of pulmonary anthrax
Spores from the dust of wool, hair, or hides are inhaled
and phagocytosed in the lungs and transported by the
lymphatic drainage to the mediastinal lymph nodes,
where germination occurs. This is followed by toxin
production and the development of hemorrhagic
mediastinitis and sepsis, which are usually rapidly fatal.
In anthrax sepsis, the number of organisms in the blood
exceeds 107/mL just before death.
 Pulmonary anthrax

Clinical findings:
The incubation period in inhalation anthrax may be as
long as 6 weeks. The early clinical manifestations are
associated with marked hemorrhagic necrosis and
edema of the mediastinum. Substernal pain may be
prominent, and there is pronounced mediastinal
widening visible on chest radiographs.
Hemorrhagic pleural effusions follow involvement of
the pleura; cough is secondary to the effects on the
trachea. Sepsis occurs, and there may be
hematogenous spread to the gastrointestinal tract,
causing bowel ulceration, or to the meninges, causing
hemorrhagic meningitis. The fatality rate in inhalation
anthrax is high in the setting of known exposure; it is
higher when the diagnosis is not initially suspected.
 Gastrointestinal:
The symptoms of gastrointestinal anthrax include
vomiting, abdominal pain and bloody diarrhea.

Injection anthrax:
It is characterized by extensive , painless, subcutaneous
edema and the notable absence of eschar characteristics of
cutaneous anthrax. Patient may progress to hemodynamic
instability due to septicemia.
 Lab diagnosis
1.Specimen collection:
• Fluid or pus from a local lesion, pleural fluid, CSF in inhalational anthrax
with sepsis.
• Stool and other intestinal contents in case of gastrointestinal anthrax.

2.Microscopic examination:
• Gram stained smears show large, gram-positive rods in chains .

• Spores are usually not seen in smears of exudate because spores form
when nutrients are insufficient and nutrients are plentiful in infected
tissue.
• Anthrax can be identified in dried smears by immunofluroscence staining
technique.
• McFadyean’s reaction:
Polypeptide capsule can be demonstrated by staining
with polychrome methylene blue stain for 30 seconds.
Capsule appears as amorphous purple material
surrounding blue bacilli . This is used for presumptive
diagnosis of anthrax in animal.
 Isolation and identification from culture

When grown on blood agar plates at 37 ̊C for overnight, the

organisms produce non-hemolytic gray to white, tencious colonies
with a rough texture and a ground glass appearance. Comma shaped
outgrowths (Medusa head, ‫״‬curled hair‫ )״‬may project from the
colony.

• Demonstration of capsule requires growth on bicarbonate

containing medium in 5-7% carbon dioxide.

• In semisolid medium, anthrax bacilli are always non- motile but

related organisms ( eg, B .cereus) exhibit motility by swarming.
 Cntnd….
Gelatin stab culture:
Gelatin is liquefied mostly at the top due to
aerobic environment and the lateral extension
of liquefaction gradually tapers towards the
bottom of the medium giving a characteristics
‫״‬Inverted fir tree‫ ״‬appearance.
Immunological tests:
• Direct immunofluroscence Ab test – For detection of capsule.
• ELISA – For detection of Ab against edema and lethal factor.
• Nucleic acid base technique: PCR For identification of toxic genes.

Treatment:
Ciprofloxacin is the drug of choice. Doxycycline is an alternative
drug. No resistant strains have been isolated clinically. Ciprofloxacin
is recommended for treatment; other agents with activity include
penicillin G, doxycycline, erythromycin, and vancomycin.
 Prevention
Pre exposure prophylaxis:
The dose schedule is 0.5 mL administered intramuscularly at 0
and 4 weeks and then at 6, 12, and 18 months followed by
annual boosters. The vaccine is available only to the U.S.
Department of Defense and to persons at risk for repeated
exposure to B. anthracis. Because the current anthrax vaccines
provide short-lived immunity and hence require repeated
vaccinations, a number of new recombinant PA vaccines (rPA)
have been developed. These novel vaccines have been shown to
be very well tolerated and highly immunogenic .
 Prevention
Post exposure prophylaxis:
 Prophylaxis with ciprofloxacin or doxycycline should
be given for 60 days, and three doses of vaccine (AVA
BioThrax)should be administered.
 Raxibacumab (Abthrax®, GlaxoSmithKline, London,
UK), a recombinant human monoclonal antibody,
was FDA approved for treatment of and prophylaxis
against inhalational anthrax in late 2012.
Anthrax immune globulin intravenous (AIGIV, Cangene Corp.
Winnipeg, Manitoba, CA) is not FDA approved but could be
made available through the Centers for Disease Control and
Prevention. AIGIV is a human polyclonal anti-serum that also
inhibits binding of PA to its receptors. Like Raxibacumab, it is
used as an adjunct to antimicrobial agents for the treatment of
severe forms of anthrax.
Others Control measure includes:

1.Disposal of animal carcasses by burning or deep burial in lime

pits.
2.Decontamination ( usually by autoclaving) of animal products.
3.Protective clothing and gloves for handling potentially infected
materials.
4.Active immunization of domestic animals with live attenuated
vaccines.
5.Contacts including veterinarians and laboratory workers at risk
should be vaccinated.
6.Incinerating animals that die of anthrax, rather than burying
them, will prevent the soil from becoming contaminated with
spores.
 Bacillus cereus
Introduction:
It is normal habitant of soil, also widely isolated from
food items such as vegetable, milk, cereals, spices,
meat and poultry.
It is an important agent of food poisoning in man.
Disease:
Bacillus cereus causes gastroenteritis( food poisoning). B. Cereus
may occasionally produce disease in immunocompromised
humans ( e.g, meningitis, endocarditis ,endophthalmitis,
conjunctivitis).

B. Cereus has also been associated with localized infections, such

as wound infections, with systemic infections including
endocarditis, catheter associated bacteremia, CNS infections,
osteomylitis and pneumonia. The presence of medical device
and IV drug use predisposes to these infections.
Transmission:
Spores on grains such as rice survive steaming
and rapid frying. The spores germinate when
rice is kept warm for many hours (e.g., reheated
fried rice). The portal of entry is the
gastrointestinal tract.
 Pathogenesis
• Bacillus cereus produces two enterotoxins. The
mode of action of one of the enterotoxins is the
same as that of cholera toxin (i.e., it adds adenosine
diphosphate ribose, a process called ADP-
ribosylation, to a G protein, which stimulates
adenylate cyclase and leads to an increased
concentration of cyclic AMP within the enterocyte).
• The mode of action of the other enterotoxin
resembles that of staphylococcal enterotoxin (i.e., it
is a superantigen).
 Clinical findings
There are two syndromes.
1. One syndrome( emetic form) has a short incubation period (4
hours) and consists primarily of nausea and vomiting, similar to
staphylococcal food poisoning. It is self-limiting, with recovery
occurring within 24 hours. It begins 1–5 hours after ingestion of a
plasmid-encoded preformed cyclic peptide (emetic toxin) in the
contaminated food products. B. cereus is a soil organism that
commonly contaminates rice. When large amounts of rice are
cooked and allowed to cool slowly, the B. cereus spores
germinate, and the vegetative cells produce the toxin during log-
phase growth or during sporulation.
(2) The other ( diarrhoeal form):
It has an incubation period of 1–24 hours and is
manifested by profuse diarrhea with abdominal pain
and cramps; fever and vomiting are uncommon. In this
syndrome, ingested spores that develop into vegetative
cells of B. cereus secrete one of three possible
enterotoxins which induce fluid accumulation and other
physiological responses in the small intestine.
Laboratory Diagnosis:
This is not usually done.
The presence of B. cereus in a pt‫י‬s stool is not sufficient
to make a diagnosis of B. cereus disease because the
bacteria may be present In normal stool specimens ,a
concentration of 105 bacteria or more per gram of food
is considered diagnostic.
Treatment:
Only symptomatic treatment is given. B.cereus is
resistant to a variety of antimicrobial agents, including
penicillin and cephalosporin .Serious non-food borne
infections should be treated with vancomycin or
clindamycin with or without an aminoglycoside.
Ciprofloxacin has been useful for the treatment of
wound infection.
Prevention:
There is no specific means of prevention. Rice should
not be kept warm for long periods.
Bacillus spore used as sterilization control:
Bacillus (Spores) Used as Sterilization Control Bacillus
species are used as biological indicators to check the
efficacy of sterilization process.
For example; ™ Geobacillus stearothermophilus
(formerly Bacillus stearothermophilus) is used for
autoclave, hydrogen peroxide gas plasma sterilizer and
liquid acetic acid sterilizer .
™Bacillus atrophaeus is used for ethylene oxide
sterilizer and dry heat sterilizer.
• Biological warfare:
It is the use of biological toxins or infectious agents such as
bacteria, viruses, insects and fungi with the intent to kill, harm or
incapacitate or plants as an act of war.

• Biological weapon:
Biological weapons are microorganisms like virus, bacteria,
fungi or other toxins that are produced and released
deliberately to cause disease and death in humans, animals or
plants.
• Bioterrorism:
It is a form of terrorism ( unlawful use of weapon
against mankind) where there is intentional and
deliberate release of biological agents ( virus, bacteria,
fungi or their toxins) to cause mass illness or death of
people, animals or plants.
 History of bioterrorism attack
• The use of biological agents as weapons is not a new concept.
They have been used since ancient time.
• ™ The first bioweapon used was the fungus Claviceps pur-
purea (rye ergot) by the Assyrians, in the sixth century BC ™.
• The plague bacilli were used in 14th century ™.
• During World War I—Anthrax was used by Germany to infect
the mules and horses of enemies.
• During World War II—Japanese forces used anthrax and
plague bacilli against prisoners.
• ™ 2001 USA World Trade Center attacks—Anthrax spores
were mailed to US media and government offices during a
terrorist attack. There were 22 cases with five deaths.
Criteria of biological agents as bioweapons
The biologic agents used as bioweapons should have the following key
features:
• Should produce high morbidity and mortality in the community .
• Potential for person-to-person spread.
• Should be of low infective dose.
• Should be highly infectious by aerosol .
• Lack of rapid diagnostic facilities.
• Effective vaccine should not be available globally.
• Potential to cause anxiety.
• Availability of pathogen and feasibility of production.
• Environmental stability- should have the potential to be "weaponized''.
 Development of bioweapon
Altering the genetic makeup of organisms has become
easier and less expensive due to accessible advanced
technologies for genetically modifying organisms. There
are possibilities of misusing such technologies for the
creation of pathogenic organisms or modification of
existing microorganisms to make them more virulent.
Various possible ways to create bioweapons include:
™
 Reconstruction of known pathogenic viruses using
information on their genetic sequences
 ™Alteration of existing bacteria to make them more
dangerous (e.g. introducing drug resistance genes) ™
 Reconstruction of microorganism that releases
harmful biochemicals within the human body and
alterations to the human host ( modification of
human microbiome,decreasing immunity).
 Role of anthrax in biological warfare
• B.anthracis is a major potential agent of bioterrorism
and biologic warfare. Bacillus is called biological
weapon. The weapon was used by bioterrorism.
• A number of nations and independent terrorist
groups have biological warfare programs.
• In September 2001, an outbreak of both inhalation
and cutaneous anthrax occurred in the United States.
The outbreak was caused by sending spores of the
organism through the mail. The countries involved
this bioterrorism are Iraq, Former Soviet Union and
Japan.
• a This resulted in 22 cases of anthrax 11 patient with
inhalation anthrax nd 11 patient with cutaneous
anthrax.5 of the patients with inhalation anthrax
died. All of the other survived.
• There was an accidental release of spores of
B.anthracis from Soviet military laboratory in 1979
causing 77 cases of inhalation anthrax with more
than 66 deaths
 Prevention and preparedness
• After the 2001 anthrax attack, the US government
has established an emergency preparedness and
response network to address possible bioterrorism in
the future. The network includes National Institutes
of Health (NIH) and the Centers for Disease Control
and Prevention (CDC) as the main bodies along with
several government agencies. It targets the following
objectives:
• Understanding the basic biology of potential
bioterrorism agents Understanding the interaction
between the human immune system and these
microorganisms
• Developing and improving drugs and vaccines that
are effective against bioterrorism agents ™
• Developing tools to quickly and accurately diagnose
diseases caused by these agents ™
• Establishing resources and biosafety laboratories to
facilitate biodefense research.

Globally various agencies are working hard to curb such

problems in future. India too is moving towards
establishment of such a network.