SPORE FORMING GRAM POSITIVE BACILLI
• Although the capsule is necessary for
- Spores: responsible for resistance to
extreme conditions
AEROBIC SPORE FORMING GRAM
POSITIVE BACILLI
I. Bacillus
- There are more than 100 species within the
genus.
- Members of the genus Bacillus are
metabolically diverse, and some species are
thermophiles that grow best at 55° C or
higher.
- The survival of Bacillus spp. in nature is
aided by the formation of spores.
II. Bacillus anthracis – Non motile
- LARGEST pathogenic bacteria
- also known as “Anthrax Bacillus”
- Virulence factors
a. D-glutamic acid Capsule: This particular
isomer of glutamic acid is resistant to
hydrolysis by host proteolytic enzymes
because it is the “unnatural” form of the
amino acid. Capsule is composed of Poly-D-
glutamic Acid
virulence, antibodies against the capsule do
not confer immunity.
b. Anthrax toxin consists of three proteins:
1. Protective antigen (PA)
2. Edema factor (EF)
3. Lethal factor (LF)
• Each of which individually is nontoxic but
which together act synergistically to produce
damaging effects.
• PA serves as a necessary binding molecule
for EF and LF, permitting their attachment
to specific receptors on the host cell’s
surface.
• EF is an adenylate cyclase, release of water
and electrolytes.
• LF is a protease that inhibit protein
synthesis.
• PA + EF = EDEMA
• PA + LF = DEATH
- Cutaneous anthrax
• A small pimple (2 to 3 days after exposure)
-> eventually ulcerates and dries, forming a
depressed black necrotic central area known
as an eschar or black eschar.
- Inhalation anthrax
• also known as Woolsorter’s disease, is
acquired when spores are inhaled into the
pulmonary parenchyma.
- Gastrointestinal anthrax
• Ingestion of the spores.
• The symptoms of gastrointestinal anthrax
include abdominal pain, nausea, anorexia,
and vomiting.
Bloody diarrhea can also occur.
- Injectional anthrax – heroin type of
drug
• serologic precipitation test which detects
• Injectional anthrax is characterized by soft
anthrax antigen
tissue infection associated with “skin
• fluid 1: filtrate of 2 grams of tissue; 5 mL
popping”.
normal saline and acetic acid
• Lack of eschar, severity of disease, and
• fluid 2: 0.5 mL of anthrax antiserum
increased mortality rate make this form
• (+) result: development of a white ring of
clinically distinct from the cutaneous form.
precipitate at the junction of 2 fluids within
- Treatment
10 minutes at Room Temperature
• Most isolates of B. anthracis are
- Laboratory diagnosis
susceptible to penicillin, but resistance can
a. Microscopy
occur in the absence of β-lactamase
• B. anthracis is a large, square-ended,
production.
gram-positive rod found singly or in chains.
• The CDC recommends that ciprofloxacin
• Gives the appearance of bamboo pole
or doxycycline be used for initial
arrangement
intravenous therapy until antimicrobial
• The spores are generally not present in
susceptibility results are known.
clinical samples.
- Caution
• Spores can be observed with a spore stain.
• Caution should always be used in working
With this technique, vegetative cells stain
with an isolate suspected of being B.
red, and the spores stain green.
anthracis.
b. Culture
• Work should be performed in a biological
• The term Medusa head/ beaten egg whites
safety cabinet, and the area should be
has been used to describe the colony s
disinfected when the work is completed.
• String of pearls on MHA with penicillin
- Ascoli Test
III. Bacillus cereus
- B. cereus is a relatively common cause of
fried rice poisoning and opportunistic
infections in susceptible hosts.
- Food poisoning caused by B. cereus takes
two forms: diarrheal & emetic
a. Diarrheal Type
• meat & poultry
• 8-16 hours (long)
• heat labile enterotoxin
b. Emetic Type
• fried rice
• 1-6 hours (short)
• heat stable enterotoxin
- Culture
• Culture of suspected food from a food
poisoning incident may be done to quantify
and isolate B.
cereus.
✓>10^5 B. cereus/gram food poisoning by
this organism is confirmed.
✓To confirm the organism as the cause of
the disease, viable counts from the stool
should also be at
least 10^5 cells per gram.
- Treatment
• Most food poisoning cases caused by B.
cereus do not require antimicrobial
treatment.
• In contrast to B. anthracis, B. cereus is
resistant to penicillin and all of the other β-
lactam antibiotics except for the
carbapenems.
• Treatment with vancomycin or
clindamycin with or without an
aminoglycoside has been successful.
- Laboratory diagnosis
• Microscopy
✓Bacillus cereus bacteria stained with
Leifson dye to display their flagella
 ANAEROBIC SPORE FORMING GRAM POSITIVE BACILLI
- Anaerobes, on the other hand, are
particularly susceptible to these toxic
derivatives of oxygen because they lack the
protective enzymes superoxide dismutase
and/or catalase, or the enzymes are present
in low concentrations.
I. Clostridium tetani
- Clostridium causing Tetanus (Spasmic
Paralysis)
- also known as Racquet Bacillus
- endospore are Round & Terminal
(drumstick/ tennis racquet appearance)
- Motile by peritrichous flagella
- Spores are highly resistant to adverse
conditions
- Iodine (1%) in water is able to kill the
spores within a few hours
- Tetanospasmin
• neurotoxin and an essential pathogenic
product
• toxic to humans and various animals when
injected parenterally, but it is not toxic by
the oral route
• causes increasing excitability of spinal
cord neurons and muscle spasm
Prevent the release and GABA and Glycine
– an inhibitory enzyme which allows the
muscle to stop the muscles to move.
- This neonate is displaying a bodily rigidity
produced by Clostridum tetani exotoxin,
called neonatal tetanus.
- Sir Charles Bell’s portrait of a soldier
dying of tetanus. The characteristic rigidity
of the body is referred to as opisthotonus
arching of the back and risus sardonicus
devil grin.
- Symptoms Trismus or Lock jaw
- DPT vaccine or DTP vaccine
• combination vaccines against three
infectious diseases in humans: diphtheria,
pertussis (whooping cough), and tetanus

- Laboratory diagnosis
a. Specimen: Wound exudates using
capillary tube
b. Gram stain is a good method for
identifying Clostridium
• Cl. tetani is a Gram positive rod, motile
with a round terminal spore giving a
drumstick appearance
c. Culture:
• On blood agar and incubated anaerobically
• Growth appears as a fine spreading film
II. Clostridium perfringens
- Causative agent of GAS GANGRENE
- also known as Gas Gangrene Bacillus
- can also cause Food poisoning
- non- motile, capsulated with Subterminal
spores
- Toxins
a. a-toxin (phospholipase C, lecithinase):
most important toxin
• Lyses RBCs, platelets, leukocytes and
endothelial cell
• Increased vascular permeability with
massive hemolysis and bleeding tissue
destruction
• Hepatic toxicity and myocardial
dysfunction
b. B-toxin
• responsible for necrotic lesions in
necrotizing enterocolitis
c. Enterotoxin
• heat labile toxin produced in colon
• for food poisoning
- Laboratory diagnosis
a. Specimen: Histological specimen or
wound exudate
• Histological specimen transferred
aseptically into a sterile screw-capped bottle
& used immediately for microscopic
examination & culture
• Specimens of exudates should be taken
from the deeper areas of the wound where
the infection seems to be most pronounced
b. Culture: Anaerobically at 37°C
• On Robertson's cooked meat medium →
blackening of meat will observed with the
production of H2S and NH3
• Exhibit Double zone of hemolysis
Target hemolysis center has complete
hemolysis outer zone is incomplete
hemolysis.
c. It ferments many carbohydrates with
acid & gas
d. Nagler reaction +
• This test is done to detect the lecithinase
activity
• Inoculated on the medium containing
human serum or egg yolk (contains lecithin)
• The plate is incubated anaerobically at
37°C for 24hrs
• Colonies of Cl. perfringens are surrounded
by zones of turbidity due to lecithinase
activity and the effect is specifically
inhibited if Cl. perfringens antiserum
containing a-antitoxin is present on the
medium.
e. It acidified litmus milk with stormy clot
production
• (+) acid with Stormy Clot Fermentation
f. Reverse CAMP test
• CAMP positive S. agalactiae (Group B
Streptococcus) is streaked in SBA & C.
perfringens is streaked perpendicular to it
• (+) “arrowhead” (enhanced) hemolysis is
seen in between = C. perfringens
LABORATORY DIAGNOSIS: LITMUS
MILK
III. Clostridium botulinum
- causes
BOTULISM (Flaccid Paralysis) -Adult
- Seven neurotoxic subtypes, labeled A-G
- First recognized and isolated in 1896 by
Van Ermengem
- Botulism is a neuroparalytic disease
• Once released into the bloodstream it
irreversibly binds to the acetylcholine
receptors in the neuromuscular junction
• It alters the mechanism for acetylcholine
release, making the neuron unresponsive to
action
potentials
- Infant Botulism: Floppy Baby Syndrome
• Most common form
• Afflicts babies from 1 week – 1 year
• Antigenic variation types A/B
• Caused by:
✓Ingesting contaminated foodstuff
✓Untreated natural honey and corn syrup
✓Household dust containing C. botulinum
spores
- Management
• Trivalent (A, B, E) antitoxin must be
administered intravenously (recovery takes
several weeks)
• Mechanical respirator is administered if
necessary.
- Laboratory diagnosis
• Toxins found in serum, leftover food.
• In infants, found in stool.
• spores are Oval, Subterminal
• Lipase Positive
IV. Clostridium difficile
- A common cause of nosocomial antibiotic-
associated diarrhea (AAD) & antibiotic
(Clindamycin)- associated
pseudomembranous colitis
- The only nosocomial organism that is
anaerobic and forms spores (survive
>5months and hard to destroy)
- Pathogenesis is mainly due to toxin
production: Infective dose is <10 spores
- Laboratory diagnosis
• specimen: Stool
• cultured on Cyclocidin-Cefoxitin Fructose
Agar = (+) colonies with Yellow Halo
- Best strategy for C.difficile testing
• For clinical use: two-step testing uses
initially EIA detection screening followed
by cytotoxicity assay or toxigenic culture for
confirmation
• Gold standard is stool culture followed by
toxigenic culture assay