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Transcript of Spore-forming gram-positive bacilli Bacillus (Aerobic) B. antheracis, B.cereus, B. subtilis...
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Spore-forming gram-positive bacilli
• Bacillus (Aerobic) B. antheracis, B.cereus, B. subtilis
• Clostridum (Anaerobic) C. tetani, C. botulinum, C. perfringens, C.
difficile
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Bacillus anthracis
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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.
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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.
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Properties• 1 - 1.2µm in width x 3 - 5µm in
length• Gram + rod• Facultative anaerobe• A large rod with square ends. • Frequently in chains
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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)
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Gram stain. The cells have characteristic
squared ends. The spores are highly refractile to light and resistant to staining.
Bacillus cereus
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Robert Koch's original micrographs of the anthrax bacillus
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Genetics
• 1 chromosome– 5.2 million bp– Ames strain
sequenced
• 2 plasmids– px01
• 184 kbp• Exotoxin
– pX02• 95.3 kbp• Capsule
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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)
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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
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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.
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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
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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
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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
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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
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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.
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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%)
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Cutaneous Anthrax (3)
CDC, Cutaneous Anthrax—Vesicle DevelopmentCDC, Cutaneous Anthrax—Vesicle Development
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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.
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• The two lungs are separated by mediastinum: Heart, trachea, esophagus, blood vessels.
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Inhalation Anthrax (2)
• Spores germinate and replicate in the lymph nodes.
• Exotoxins: mediastinal widening and pleural effusions (accumulation of fluid in the pleural space).
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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%)
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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.
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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.
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This picture is 9 days after the onset of symptoms of oral-pharyngeal anthrax, an
unusual manifestation of humaninfection with B. anthracis.
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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
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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.
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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
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2nd Phase Symptoms
• Breathing problems, pneumonia
• Shock
• Swollen lymph glands
• Profuse sweating
• Cyanosis (skin turns blue)
• Death
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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
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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
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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
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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).
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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
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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
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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
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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
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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
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Weaponization & Weaponization & Bacillus Bacillus AnthracisAnthracis::
Why is this Agent Considered to be the Department of Defense’s Number - One/Two Biological Threat?
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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
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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)
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How to Aerosolize?
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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
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Bacillus cereus
• Motile• No capsule• Saprophyte
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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)
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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.
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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.
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Lab. diagnosisLab. diagnosis• Not usually done
TreatmentNo antibiotic is given. Only symptomatic
treatment
PreventionGrains (specially rice) should not be reheated