GENIO, EMMANUEL JOHN A.

BSMT L3

GENUS BACILLUS
General characteristics of Genus Bacillus

Epidemiology Other significant characteristics Laboratory diagnosis

Can be isolated from soil from all climates, thermal springs, fresh and saltwater Survival in nature is due to endospores Gram Stain: spores appear only as empty spaces, not stained
and plant materials
ENDOSPORES Facultative anaerobic
● Highly resistant to heat and desiccation
● Spores remain viable in a dormant state until they are deposited Under aerobic conditions form endospores
in a suitable environment for growth(moisture, temperature,
oxygenation, nutrient availability Catalase (+)
● Has the ability to survive harsh environments
○ Infectiousness
○ Ease of aerosol dissemination
○ High mortality rate
■ EFFECTIVELY USED AS AN AGENT OF
BIOLOGIC WARFARE

THREE GROUPS OF ENDOSPORES BASED ON LOCATION AND SIZE

GROUP I GROUP ll GROUP lll

Spores are oval or cylindrical, central or terminal, sporangia: not swollen Spores are oval, central or terminal, sporangia: swollen Spores are round, terminal swollen sporangia
 GENIO, EMMANUEL JOHN A.
BSMT L3

BACILLUS ANTHRACIS
Morphology Survival in soil Laboratory diagnosis

Samples for diagnosis Specimen treatment Animal inoculation test

Large square-ended Spores remain viable in soil Samples are collected depending on Heat or Alcohol Shock is a necessary prior to plating on solid Animal inoculation test: Experimental animals are injected
gram-positive rod, singly for decades. the site affected: media, an enrichment and selection procedure to enhance intraperitoneally by a suspension of the test organism
or in chains( if in chains ● Swab samples from cutaneous recovery and isolation of Bacillus species "Suspected B. anthracis culture"
appear as bamboo rods) In World War II in Scotland lesions and blood cultures ● The animal dies in 48-96 hours due to respiratory failure
spores were exploded. ● Sputum and blood for Pre-treatment removes contaminating organisms, only ● Large number of typical bacilli can be found in the blood
Non-motile pulmonary anthrax spore-forming bacilli survives and tissue of spleen of the infected animal
Survived for >40 years and ● Gastric aspirate, feces and
Capsule could be were eradicated in 1987 blood for enteric anthrax Heat shock treatment can be utilized for the growth and
demonstrated during growth enhancement of endospores from clinical specimens
in infected animals. Changing environmental
conditions (temp. rain etc.) 70°C for 30’ or 80°C for 10’ for killing vegetative cells and
Spores are ellipsoidal to help in survival and retaining spores for most Bacillus species
cylindrical/oval and multiplication.
centrally located, not 62°C to 65°C for 15’ – 20’ for Bacillus anthracis
swollen
Direct detection methods Cultural characteristics

GRAM STAINING SPORE STAINING BAP PLET AGAR Bicarbonate agar

Only specific procedure for direct
detection of Bacillus species
Appear as large gram (+) rods in
singles, pairs or serpentine changes
Gram stain of Bacillus anthracis
showing Gram positive rods forming
long filaments. “Bamboo–like”
appearance
Smear is covered with
Malachite Green
Filter paper placed over the
stain
Slide is heated for several
minutes to force the dye into
the cell walls of the spore
Keep filter paper moist so that
the stain is steamed rather than
baked into the endospores
Safranin counterstain follows
the primary stain
medium to large grayish to Polymyxin-lysozyme-EDTA-tha Used to induce capsule formation,
white colonies, flat, irregular llous acetate Agar presumptive morphologic identification
with swirling projections,
MEDUSA HEAD or COMET Creamy white, domed,
TAIL, ground glass appearance, circular colonies
nonhemolytic
Colonial growth of Bacillus
anthracis. When lifted by an
inoculation loop, colonies, in
this case on sheep blood
agar, show a tenacity that
allows them to be pulled up
and stay upright with a
texture similar to egg whites.

ENDOSPORE: GREEN
VEGETATIVE CELLS: PINK

Biochemical test reactions

Other biochemical tests Gelatine liquefaction test Lysis by gamma phages String of Pearl Test AST

Virulence factor
ANTHRAX TOXIN: composed of three proteins
● Protective Antigen (PA)
○ Facilitates transport of EF and LF
● Edema Factor (EF)
● Lethal Factor (LF)
○ Responsible for death
● Carbohydrate fermentation test:
Positive
● Nitrate reduction test: Positive
● Starch hydrolysis test: Positive
● Voges-Proskauer test: Positive
Treatment
Penicillin is the drug of choice.
For penicillin-sensitive patients:
tetracycline, erythromycin,
chloramphenicol, and streptomycin
be given as alternative drugs.
Slow liquefaction Growth has This test accurately
a characteristic appearance of differentiates B.anthracis
an inverted pine tree. from other bacillus species.
Lysis by gamma phages:
Positive
Lysis of Bacillus anthracis by
the lytic phage gamma. The
plaque (clear area) in the region
of confluent growth is where the
gamma phage was applied.
The plaque results from the
phage's
Pathogenesis
CUTANEOUS ANTHRAX/ BLACK ESCHAR / MALIGNANT
PUSTULE
● 95-98% of anthrax cases are of this type.
● Infection occurs through wounds, burns, cuts, abrasions,
or insect bites contaminated with spores which may
progress to toxemia and septicemia.
● The site of entry often produces a painless blister
referred to as Malignant pustule.
GENIO, EMMANUEL JOHN A.
Inoculate the suspected isolate
onto an agar containing
penicillin
Incubate for 3 – 6 hours at
37°C
Area of inoculation are
examined microscopically for
the presence of large spherical
bacilli in chains
String of Pearl Test: Positive
ENTERIC "INTESTINAL"
ANTHRAX
● Caused by the
ingestion of spores
from infected meat
and inoculated into a
lesion on the intestinal
mucosa
BSMT L3
Sensitive to penicillin
PULMONARY ANTHRAX
● Caused by the inhalation
of a large number of B.
anthracis spores.
● It is usually fatal.
● This clinical form is
commonly known as
"wool sorter disease".
○ Spores are inhaled
during shearing,
sorting, or handling
of animal hair
○ Large numbers of
bacilli are found in
sputum
 GENIO, EMMANUEL JOHN A.
BSMT L3

BACILLUS CEREUS
General characteristics Pathogenicity Culture medium Other bacillus species
● Very close relative of B. anthracis DIARRHEAL MYEP or MYP ● Bacillus subtilis – Hay’s Bacillus
● Beta-hemolytic ● ingestion of meat or poultry, 8 – 16 hours following ● Mannitol, Egg Yolk and Polymixin B Agar ● Bacillus steathermophilus – spores are
● Penicillin resistant ingestion used to evaluate efficacy of autoclave
● Motile ○ Toxins: hemolysin BL (Hbl), nonhemolytic PEMBA ● Bacillus thuringiensis
● Found also within soil enterotoxin (Nhe), cytotoxin K (CytK) ● Polymixin B, Egg Yolk, Mannitol, Bromthymol Blue ● Bacillus mycoides
● Opportunistic pathogen ● Bacillus circulans
● Associated with foodborne illness (meat, EMETIC BCM ● Bacillus licheniformis - SOURCE OF
vegetables, deserts, sauces, milk ● ingestion of fried rice, 1 - 5 hours following ingestion ● Bacillus cereus Medium ANTIBIOTIC
● Higher incidence is seen following ingestion ○ Heat-stable, proteolysis and acid-resistant ● Bacillus megaterium
of rice dishes toxin: CEREULIDE produced in food ● Bacillus clausii - used to treat diarrhea
● Bacillus coagulans
● Bacillus pumilus
● Bacillus sphaericus

Comparison between B.cereus and B.anthracis

Characteristics Bacillus anthracis Bacillus cereus

Penicillin susceptibility susceptible Not susceptible

Growth requirement for thiamin + -

Hemolysis on sheep blood agar - +

Glutamyl-polypeptide capsule + -

Lysis bt gamma phage + -

motility - +

Growth on chloral hydrate agar - +

String of pearl test + -

GENUS PAENIBACILLUS and GENUS BREVIBACILLUS

General characteristics Other species
Rare cause of human infection ● Brevibacillus brevis
● Paenibacillus macerans
Common environmental contaminants ● Paenibacillus alvei
● Paenibacillus polymyxa
ID not recommended unless isolated in sterile sites or found in large numbers in pure culture
 GENIO, EMMANUEL JOHN A.
BSMT L3

Prevention
Cell-free inactivated vaccine given in five doses (0 weeks, 4 weeks, 6 months, 12 months, 18 months) with annual boosters thereafter for high risk adults

Chemoprophylaxis with ciprofloxacin or doxycycline for a minimum of 4 weeks is recommended following aerosol exposure to Bacillus anthracis as may follow a bioterrorist event

Disinfection
Disinfectants used prior to disposal of suspected specimens containing large number of spores:
● Formaldehyde
● Glutaraldehyde
● Hydrogen Peroxide and Peracetic Acid