Transcript Bacillus anthracis
Bacterial agents of bioterroism
Laboratory network for biological terrorism
Bacillus anthracis
Anthrax
Anthrax: Overview • Primarily disease of
herbivores
• Humans usually
infected by contact with infected animals or contaminated animal products
• Soil reservoir • Woolsorter’s disease
(inhalation anthrax)
• No person-to-person
transmission of inhalational anthrax
CDC
ANTHRAX • Three forms of human anthrax occur: 1. Cutaneous 2. Gastrointestinal • Oropharyngeal • Abdominal 3. Inhalation (Woolsorter’s Disease)
Cutaneous anthrax
Vesicle development, day 2 Eschar formation, day 4
Inhalation Anthrax • Infective dose = 8,000 - 15,000 spores • Incubation period = 1-6 days • Duration of illness = 3-5 days • Fever, malaise, and fatigue • Short period of improvement = up to 2 days • Abrupt respiratory distress…death <24hrs • Person to person transmission = no
Anthrax: Specimen Selection • Inhalation: Sputum and Blood • Cutaneous: Vesicles and Eschar • Gastrointestinal: Stool and Blood
Bacillus anthracis Key Sentinel Lab Tests • Gram stain • Growth characteristics on agar • Sporulation, in air • Motility • Capsule by India Ink
Bacillus anthracis Gram Stain Morphology
• Broad gram-positive rod: 1-1.5 X 3-5 µ • Oval, central - subterminal spores: 1 X 1.5 µ
with no significant swelling of cell
• Spores are NOT usually present in clinical
specimens unless exposed to atmospheric O 2
B. anthracis,
Gram stain demonstrating spores
B. anthracis Colonial Morphology
• Colonial morphology of 18-24hr @ 35 C: – Well isolated colonies are 2-5 mm in diameter – Flat or slightly convex, irregularly round – Edges: slightly undulate, often curly tailing
edges
– Ground glass appearance – “Sticky” consistency….stands up like beaten
egg whites
B. anthracis
, colony on SBA
“STICKY” consistency of anthracis’ colony on SBA B.
Bacillus anthracis Presumptive Identification
• Gram-positive, broad rod, catalase-
positive, spore-positive, aerobe: Bacillus sp.
• Spores are oval and nonswelling with
ground glass colony appearance: Bacillus morphology group 1, includes B. anthracis, B. cereus, B cereus var mycoides, and B. thuringiensis
Bacillus anthracis Presumptive Identification, con’t
• Nonmotile:
B anthracis and B cereus var mycoides ( megaterium) and B.
• Nonhemolytic, forms capsule:
Presumptive B. anthracis
• Refer to state lab for testing
Yersinia pestis
Plague
Plague: Overview
• Natural vector - Rodent flea • Mammalian hosts – rats, squirrels,
chipmunks, rabbits, and carnivores
• Enzootic or Epizootic
Plague Epidemiology • U.S. averages 13 cases/yr • 30% of cases are in Native Americans in the
Southwest. 15% case fatality rate
• Most cases occur in summer and near the
patient’s residence
– bubonic (infected lymph nodes) – septicemic (blood-borne organisms) – pneumonic (transmissible by aerosol;
deadliest)
Yersinia pestis
Specimen Selection • Specimen selection is important – Bubo - lymph node aspirate – Blood - organisms may be intermittent. Take
three specimens 10-30 minutes apart
– Pneumonic • Sputum/throat - use Wayson stain • Bronchial washings - Wayson stain • Inoculate routine plating media
Sentinel Lab Procedures
Yersinia pestis
• Gram stain • Wayson stain • Growth characteristics on agar • Growth characteristics in broth
Yersinia pestis
Gram stain • Small, gram-negative coccobacilli
Yersinia pestis
Wayson Stain • Used for rapid assessment – when it is a part of the identification process • Best with tissue, sputum, blood • Stains of pure culture isolates tend to lose bipolarity • Pink-blue cells with polar granules (safety pin appearance)
Yersinia pestis
Wayson Stain • Pink-blue cells with a closed safety pin look
Wayson stain alone is not diagnostic
Y.pestis
48 h culture on SBA 72 h culture on SBA
Yersinia pestis Technical Hints
• Small Gram-negative, poorly staining rods
from blood, lymph node aspirate, or respiratory specimens
• Safety pin appearance in Gram, Wright,
Giemsa, or Wayson stain
• More than one patient in a short, specified
period with fever, lymphadenopathy
• Refer to state lab
Francisella tularensis Tularemia
Tularemia: Overview • Disease of Northern Hemisphere • In U.S., most cases associated with rabbits/hares and ticks • About 200 cases/year in U.S.
– most in South central and Western states – majority of cases in summer, some in winter
Reported Cases of Tularemia 1990-1998
Tularemia: Overview (cont’d) • Several forms of human tularemia exist: - Ulceroglandular, glandular, oculoglandular, oropharyngeal, intestinal, pneumonic, and typhoidal • Low infectious dose –1 to 10 organisms by aerosol or intradermal
route
• No person-to-person transmission
Tularemia: Specimen Selection • Serum - acute and convalescent • Blood cultures • Sputum • Swab – ulcer or eye
Sentinel Lab Procedures
Francisella tularensis
• This is a dangerous, highly virulent organism and it should not be manipulated at the bench. Laboratory-acquired infections can occur easily .
• Gram stain • Growth characteristics in broth • Growth characteristics in agar
Francisella tularensis
• Poorly staining, tiny Gram-negative coccobacilli
Francisella tularensis
Growth Characteristics • Fastidious, requires cysteine for robust growth: Cysteine Heart Agar (CHA) is ideal – Enriched chocolate agar + 9% sheep blood + cysteine – Not part of Sentinel Lab routine procedures – BCYE (for
Legionella ) also works
• Will grow initially on sheep and chocolate blood agar and Thayer-Martin agar, but poorly or not at all on passage • Grows slowly at 35 o C, poorly at 28 o C
Francisella tularensis
Growth Characteristics • 24 hours – gray-white, translucent colonies – usually too small to be seen individually • 48 hours – Sheep Blood Agar - <1 mm, gray-white, opaque, no
hemolysis
Francisella tularensis
Sheep blood agar Chocolate agar Cysteine heart agar
Francisella tularensis
Technical Hints
If you see:
• Tiny, Gram-negative coccobacilli from
blood, lymph node aspirate, or respiratory specimens
• Blood isolates that will grow slowly on
chocolate agar but poorly or not at all on blood agar in 24 hours
• Faint growth in thio; requires cysteine in
other broth
• Refer to state lab
Brucella spp.
Brucellosis
BRUCELLOSIS • A zoonotic disease caused by any of 4
Brucella and canis sp.: abortus, melitensis, suis,
• A systemic infection characterized by
an undulant fever pattern
• But relatively rare in the U.S. with
approximately 100 cases/yr
BRUCELLOSIS: TRANSMISSION • Unpasteurized dairy products – The most common mode of transmission • Direct skin contact – Occupational hazard for farmers,
butchers, veterinarians, and laboratory personnel
• Aerosols – Highly infectious
BRUCELLOSIS
•Infective dose = 10 -100 organisms •Incubation period = 5 days - > 6 months •Duration of illness = weeks to months •Fever, profuse sweating, malaise, headache and muscle/back pain. •Person to person transmission = no •Mortality = <5% •Persistence of organism = very stable
Brucella
spp.
Specimen Selection • Serum – The diagnosis of brucellosis is frequently
achieved by serology. An acute & convalescent phase specimen should be collected (21d apart)
• Blood or bone marrow – Sources from which Brucellae are most
often isolated
• Tissue (spleen, liver) – Brucellae occasionally isolated
Brucella spp.
Biosafety Alert
•
Brucellosis
is THE most commonly
reported laboratory-associated bacterial infection.
• Cases have occurred in clinical
laboratory settings by “sniffing” cultures, direct skin contact with cultures, and aerosol generating procedures
Sentinel Lab Tests Brucella spp.
• Colonial morphology on SBA • Gram stain morphology • Oxidase positive • Urea hydrolysis positive
Brucella spp.
Key Sentinel Lab Tests
Colonial morphology on SBA
–Fastidious –Visible growth may take 48 - 72 hrs –Small (0.5-1.0mm), convex, glistening – Non-hemolytic and non-pigmented
B. melitensis on sheep blood agar
Brucella spp.
Key Sentinel Lab Tests
Gram Stain Morphology
–Tiny (very) –Faintly staining –Gram-negative coccobacilli –0.5 - 0.7
x 0.6 - 1.5
Brucella spp.
Review of Key Tests
• Tiny, faintly staining, gram-negative coccobacilli from blood or bone marrow • Slow growth on Sheep Blood Agar, 2-3 days for colony appearance • Oxidase + • Urease + • Handle plates with care • Refer to state lab
Clostridium botulinum
Botulism
Botulism: Overview
• Caused by toxin from – toxin types A, B, E, most commonly associated with
human disease
– most potent lethal substance known to man (lethal
dose 1ng/kg)
Clostridium botulinum • C. botulinum spores found in soil worldwide • Approximately 100 reported cases/year in the U.S.
– infant most common (72%) – food borne not common • No person-to-person transmission
FOODBORNE BOTULISM
• Infective dose: 0.001 g/kg • Incubation period: 18 - 36 hr (6hr to 10 d) • Dry mouth, double vision, droopy eyelids, dilated pupils • Generalized, progressive descending bilateral muscle weakness & paralysis • Respiratory failure and death • Mortality usually 5 – 10%
FOODBORNE BOTULISM
• Among 309 persons with clinically diagnosed botulism reported to CDC from 1975 to 1988: – Stool cultures for
C. botulinum : 51% +
– Serum botulinum toxin testing: 37% + – Stool botulinum toxin testing: 23% + • Overall, at least one of the above tests was positive for 65% of all patients • Diagnosis is primarily clinical
Sentinel Lab Procedures for Botulism Event
• Properly collected specimens are to be referred to designated testing laboratories • Prior to the shipment of any botulism associated specimen, testing must be arranged with MDCH laboratory
Sentinel Lab Procedures for Botulism Event Clinical specimens to be collected:
1. Serum 2. Feces 3. Food samples
Autopsy specimens:
1. Serum 2. Gastric and intestinal contents
Botulism Biosafety Alert
Materials suspected of containing botulism toxin must be handled: –Biological Safety Cabinet (Class II) –Laboratory Coats –Disposable surgical gloves –Face shield (as needed)
BOTULISM
• The diagnosis of botulism is made
clinically, i.e., based on the patient’s case history and physical findings
• Health care providers suspecting
botulism should contact the Michigan Department of Community Health
Botulism Referral Lab Procedures • Mouse bioassay • Isolation of C. botulinum