Spore-forming gram-positive bacilli

• Bacillus (Aerobic) B. antheracis, B.cereus, B. subtilis

• Clostridum (Anaerobic) C. tetani, C. botulinum, C. perfringens, C.

difficile

Bacillus anthracis

Bacillus anthracis

• DiseaseAnthrax (common in animal but rare in humans).

• 1876 - German bacteriologist Robert Koch confirmed bacterial origin of anthrax.

• 1945 - In Iran an anthrax outbreak killed more than 1 million sheep.

The anthrax bacillus, Bacillus anthracis, was the first bacterium shown to be the cause of a disease.

History (Recent years)

• 1991 - About 150,000 U.S. troops were vaccinated for anthrax in preparation for Persian Gulf War.

• 2001 - Letters containing anthrax spores were mailed to many places in the US such as NBC, New York Times, and Media in Miami.

Properties• 1 - 1.2µm in width x 3 - 5µm in

length• Gram + rod• Facultative anaerobe• A large rod with square ends. • Frequently in chains

PropertiesoA 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)

Gram stain. The cells have characteristic

squared ends. The spores are highly refractile to light and resistant to staining.

Bacillus cereus

Robert Koch's original micrographs of the anthrax bacillus

Genetics

• 1 chromosome– 5.2 million bp– Ames strain

sequenced

• 2 plasmids– px01

• 184 kbp• Exotoxin

– pX02• 95.3 kbp• Capsule

Anthrax

• From the Greek word From the Greek word anthrakosanthrakos for coal for coal• Caused by sporesCaused by spores• Primarily a disease of Primarily a disease of domesticated & wild animalsdomesticated & wild animals

– Herbivores such as sheep, cows, horses, goatsHerbivores such as sheep, cows, horses, goats• Natural reservoir is Natural reservoir is soilsoil

– Does not depend on an animal reservoir making it Does not depend on an animal reservoir making it hard to eradicatehard to eradicate

– Cannot be regularly cultivated from soils where Cannot be regularly cultivated from soils where there is an absence of endemic anthrax there is an absence of endemic anthrax

• Anthrax zonesAnthrax zones– Soil rich in organic matter (pH < 6.0) Soil rich in organic matter (pH < 6.0)

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

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.

Three forms of Anthrax

• CutaneousCutaneous anthrax anthrax– Skin Skin – Most commonMost common– Spores enter to skin through small lesionsSpores enter to skin through small lesions

• InhalationInhalation anthraxanthrax– Spores are inhaledSpores are inhaled– The most lethal type of Anthrax.

• GastrointestinalGastrointestinal (GI) (GI) anthraxanthrax– Spores are ingestedSpores are ingested– Oral-pharyngeal and abdominalOral-pharyngeal and abdominal

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

Capsule

Sticky, gelatinous polymerpX02 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

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

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.

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%)

Cutaneous Anthrax (3)

CDC, Cutaneous Anthrax—Vesicle DevelopmentCDC, Cutaneous Anthrax—Vesicle Development

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.

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

Inhalation Anthrax (2)

• Spores germinate and replicate in the lymph nodes.

• Exotoxins: mediastinal widening and pleural effusions (accumulation of fluid in the pleural space).

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%)

Gastrointestinal Anthrax

o GI anthrax may follow after the consumption of contaminated, poorly cooked meat.

o There are 2 different forms of GI anthrax:

1) Oral-pharyngeal 2) Abdominal

o Abdominal anthrax is more common than the oral-pharyngeal form.

GI Anthrax (2)o Oral-pharyngeal form - results from the deposition and

germination of spores in the upper gastrointestinal tract.

o Local lumphadenopathy (an infection of the lymph glands and lymph channels), edema, sepsis develop after an oral or esophageal ulcer.

o Abdominal form - develops from the deposition and germination of spores in the lower gastrointestinal tract, which results in a primary intestinal lesion.

o Symptoms such as abdominal pain and vomiting appear within a few days after ingestion.

This picture is 9 days after the onset of symptoms of oral-pharyngeal anthrax, an

unusual manifestation of humaninfection with B. anthracis.

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

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.

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

2nd Phase Symptoms

• Breathing problems, pneumonia

• Shock

• Swollen lymph glands

• Profuse sweating

• Cyanosis (skin turns blue)

• Death

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

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

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.

At day 1At day 1 At day 3At day 3

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).

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

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

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

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

Treatmento Penicillin (Before 2001, 1st line of treatment

was penicillin G but Stopped for fear of genetically engineered resistant strains)o Doxycyclineo Ciprofloxacin (from fluoroquinolones)

o

Weaponization & Weaponization & Bacillus Bacillus AnthracisAnthracis::

Why is this Agent Considered to be the Department of Defense’s Number - One/Two Biological Threat?

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

Specific Benefits of Using Anthrax as a Specific Benefits of Using Anthrax as a Biological Weapon 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)

How to Aerosolize?

Protection Against AnthraxProtection 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

Bacillus cereus

• Motile• No capsule• Saprophyte

Laboratory differentiation of Bacillus anthracis & B. cereus

Characteristics B. anthracis B.cereus

Hemolysis onBAP

= +

Motility = +

String of pearls + =

Growth on PEA = +

Gelatin hydrolysis = +

Susceptibility toPenicillin (10U/ml)

Susceptible Resistant

(phenethyl alcohol)

Bacillus cereusBacillus cereus• Disease Food poisoningRare infections: Meningitis, Osteomyelitis, …

• TransmissionSpores on grains survive during steaming and rapid

frying. Spore germinated when rice is reheated.Portal of entry is the gastrointestinal tract.

PathogenesisPathogenesis

• B. cereus produces 2 enterotoxins.

Clinical findingsClinical findings1.Emetic syndromeA short incubation period (4 hours) with nausea and

vomiting similar to staphylococcal food poisoning.

2.Diarrheal syndromeInvolves a long incubation period (18 hours) with

diarrhea and resembles clostridial gastroenteritis.

Lab. diagnosisLab. diagnosis• Not usually done

TreatmentNo antibiotic is given. Only symptomatic

treatment

PreventionGrains (specially rice) should not be reheated