CLINICAL BACTERIOLOGY (LECTURE)

LESSON 11: SPORE-FORMING, CATALASE-POSITIVE BACILLI

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2nd SEMESTER I S.Y. 2021-2022
TRANCRIBED BY: JEAN HERSHEY REYES

Bacillus Inhalation Anthrax

 Gram positive or gram variable

 Aerobic or facultative anaerobic bacilli  Also called wool-
 Spore former- in order to harsh environments or lack of sorter’s disease,
nutrients acquired through
 Catalase Positive inhalation of spores
 Motile, except for B. anthracis  2-5 days after
 In contrast with the genus Clostridium, Bacillus form spores exposure, patient
aerobically and are catalase-positive may manifest with
mild fever, fatigue,
and malaise
Bacillus anthracis (Anthrax bacillus)
(resembles flu)

 This mild form of the disease lasts 2-3 days, which is

 The causative agent of followed by a sudden severe phase characterized by
anthrax respiratory problems followed by disorientation, coma, and
 Virulence factor: glutamic death; shortness of breath and cyanosis due to the lack of
acid capsule and anthrax oxygen in the blood
toxin
 The toxin is composed of 3
components: protective Gastrointestinal Anthrax
antigen (PA), edema factor
(EF) and lethal factor (LF)
 Ingestion of spores
 PA + EF = edema
 Symptoms include abdominal pain, nausea, anorexia, and
 PA + LF = death of host cells
vomiting; bloody diarrhea
 The fatality rate is higher than cutaneous anthrax (it is hard to
Clinical Infection
diagnose)
o Anthrax is a common disease of live-stock
o Woolsorter’s disease or ragpicker’s disease
o Human infection is acquired from exposure to animals or Injectional Anthrax
animal products
o Four forms are seen in humans: cutaneous (skin),
inhalation/pulmonary (lungs and respiratory tract),  A new form characterized by soft tissue infection associated
gastrointestinal (intestine), and injectional anthrax (soft tissue) with injection of drugs of abuse; direct inoculation of
spores in the tissue
o Injectional anthrax emerged recently, resulting from the  First recognized in Scotland during an outbreak in 2009-2010
injection of spores into tissue during administration of drugs (heroin use); much higher mortality rate
of abuse  This form can be associated with necrotizing fasciitis, organ
failure, shock, coma, and meningitis
 It is not associated with eschar formation (lack)
Cutaneous Anthrax

Complications
 Occurs when wounds
are contaminated
with spores acquired  Approximately 5% of patients with anthrax develop
through skin cuts, meningitis, especially those with inhalation and injectional
abrasions, or insect forms
bites  Unconsciousness and death may occur following 1 to 6 days
 Starts as a small after exposure
pimple or papule at the
site of inoculation 2-3
days after exposure Laboratory Diagnosis (B. anthracis)
 An erythematous ring develops, and a small dark area appears Specimen
in the center of the ring and eventually ulcerates and dries  Malignant pustule, sputum, and blood
(eschar or black eschar)- 1-2 weeks  B. anthracis is typically isolated from normally sterile
 The lesion is also known as malignant pustule sites such as blood, lung tissue and CSF

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Microscopy around colonies on egg-yolk agar
 B. anthracis is large (1.0 to 1.5 um x 3.0 to 5.0 um),  Grows in high salt concentration (7% NaCl) and low
square-ended, gram positive or gram variable rod found pH (<6)
single or in chains  Susceptible to penicillin
 When in chains, the cells give the appearance of  Capsule can be detected using the India ink stain on
bamboo rods/poles blood or CSF
 In gram stain preparations of clinical samples, vegetative
cells can appear with clear zones (presence of
capsule) around the cells
 Spores can be seen as unstained areas within the cells
 Using a spore stain, the vegetative cells stain red and
the spores stain green structures

Bamboo rod appearance Spore stain

with unstained spores

 B. anthracis can be
inoculated onto
agar containing
penicillin
 After incubation for
3-6 hours at 37
degrees, the areas
of inoculation are
examined
microscopically for presence of large spherical bacilli in
chains – “string of pearls” appearance Bacillus cereus (Fried rice bacillus)

Culture
 Culture media for  B. cereus is a relatively common cause of food poisoning
isolation: Blood agar and opportunistic infection in susceptible host
plate, PEA for stool
 Two forms of food poisoning: diarrheal and emetic (self-
specimen
limiting)
 On blood agar, colonies
 Eye infections – endophthalmitis, keratitis with abscess
appear nonhemolytic,
formation
large (2-5 mm), gray, and
flat with an irregular  Also implicated in cases of meningitis, septicemia, endocarditis,
margin because of osteomyelitis
outgrowth of long filamentous projections – “medusa  Non-gastrointestinal infections occur more frequently in IV drug
head” colonies; irregular margin users, neonates, immunosuppressed and postsurgical patients

 Colonies have tenacious Diarrheal Form

consistency, holding tightly to
the agar surface, and when the o From ingestion of meat or poultry, vegetables, and pasta
edges are lifted with a loop, o Incubation: 8-16 hours (long incubation)
they stand upright without o Characterized by abdominal pain and diarrhea, and about
support 25% of individuals have vomiting
 Described as having the o Duration is about 24 hours
appearance or characteristic of o Caused by a heat labile enterotoxin
beaten egg whites
 “Inverted fir tree” appearance Emetic Form
in gelatin medium;
o The predominant symptoms is nausea and vomiting 1 to 5
hours (short incubation) after ingestion of contaminated food
o Diarrhea is present only in 1/3 of affected individuals
Test for Identification o Associated with ingestion of fried rice
 Catalase test positive, grows aerobically or o Average duration is about 9 hours
anaerobically, and is nonmotile (motility media) o Caused by a heat stable enterotoxin
 Ferments glucose, but not mannitol, arabinose, or
xylose
 Lecithinase positive – opaque zone can be seen

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 Laboratory Diagnosis (B. cereus) o Type A: mild and self-limiting GI illness
Specimen  After 8-30 hour incubation, the patient experiences
 The specimen is the suspected food diarrhea and cramping abdominal pain for 24 hours;
 Used to isolate and quantify B. cereus self-limiting
 If >105 B. cereus cells per gram of food are present and
other pathogens are absent, food poisoning by this o Type C (enteritis necroticans): more serious
organism is confirmed  After incubation of 5-6 hours, acute onset of severe
abdominal pain and bloody diarrhea occurs, and may
Culture be accompanied by vomiting
 Grows aerobically on  This is followed by necrotic inflammation of the small
blood agar at 37 intestine that may lead to bowel perforation
degrees
 B-hemolytic, frosted
glass-appearing Gas gangrene (Myonecrosis)
colonies containing
spore-forming, gram- o Occurs when organisms contaminate wounds through
positive bacilli trauma
 Motile and o C. perfringens is the most common cause, releasing potent
lecithinase positive exotoxin
o The gas gangrene exotoxins (alpha-toxin) cause necrosis of
the tissue
o Manifestations include pain and swelling in the affected
Bacillus subtilis (Hay bacillus) area
o Bullae (fluid filled blisters), serous discharge, discoloration,
and tissue necrosis are observed
 Most commonly encountered laboratory contaminant
Clostridium botulinum (Canned food bacillus)
 The source of bacitracin antibiotic
 Can cause eye infection
o Subterminal spores
 BAP: Large, flat, dull with a ground glass appearance; may be
o Virulence factor: botulism toxin, one of the most potent
B-hemolytic and may exhibit pigmentations (pink, yellow,
natural toxins known to man
orange, or brown)
o BAP – B-hemolytic colonies

Spore-Forming, Anaerobic Bacilli
Clostridium
o Anaerobic spore-forming bacilli, collectively called clostridia
o Catalase-negative, gram-positive bacilli
o Motile (except C. perfringens, C. ramosum, and C. inoculum)
o Non-encapsulated except C. perfringens
o Clostridia can be classified according to location of the
spores in the cell: terminal, subterminal, and central spores
Clinical Infection
 Bacteria or their toxins usually enters the body through
ingestion or open wounds that have been contaminated
with soil
 Diseases: tetanus, gas gangrene (myonecrosis), botulism,
and food poisoning
 In tetanus, gas gangrene, and wound botulism, spores
enter through wounds and germinate in vivo
Botulism
o Foodborne botulism results from ingestion of preformed
toxin, produced by C. botulinum in the food
o Botulinum toxin is an extremely potent neurotoxin, even a
small amount is capable of causing paralysis and death
o The toxin attaches to the neuromascular junction of nerves
and prevents the release of acetylcholine, which results in
flaccid paralysis and death
o Food sources: home-canned vegetables, home-cured meat,
fermented fish, and other preserved foods
o Clinical manifestations develop as early as 2 hours or as late
as 3-8 days
o Toxin is absorbed through the intestine and enters systemic
circulation to reach the nervous system
o Characterized by weakness and paralysis, double or blurred
vision, impaired speech, and difficulty swallowing. Respiratory
paralysis may also occur
o Treatment: antitoxin and supportive care

Clostridium perfringens (Gas gangrene bacillus) Infant Botulism Wound Botulism

o Most commonly isolated clostridium in blood cultures  Infant botulism follows  Contamination of wounds
o Virulence factor: alpha-toxin (necrosis) and enterotoxin ingestion of C. botulinum with the spores of C.
(gastrointestinal illness) spores botulinum, which
o Subterminal spores  Contaminated honey is germinates; the
o Lecithinase positive most implicated with vegetative cells
infant botulism multiple and produce
o BAP: dome-shaped  The spores germinated toxins
and grayish white with and the vegetative cells  Clinical manifestations
double zones of colonize the colon and are similar to
hemolysis (inner zone produce toxins foodborne botulism
– beta-hemolysis; outer zone – alpha-hemolysis)
 Aka flappy baby
o Can cause food poisoning and gas gangrene syndrome
(myonecrosis)
o Two types of food poisoning: Type A and Type C

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 Clostridium tetani (Tack head bacillus)

o Soil and environmental inhabitant

o Spores are found in the soil, dust, and feces of farm animals
o Virulence factor: tetanospasmin (neurotoxin)-paralysis
o BAP – Slow, anaerobic, heavy, smooth, and swarming growth
and have a matte surface with a narrow zone of B-hemolysis

o (+) gelatinase and indole; (-)

lecithinase

o Cells are with terminal spores that

have a “drumstick” or “tennis-
racket” appearance

Tetanus

o A disease attributed to the neurotoxin tetanospasmin

produced by C. tetani
o This causes spastic type of paralysis, with continuous
muscular spasm leading to trismus (lockjaw), risus sardonicus
(distorted grin), and difficulty breathing
o Occurs when spores enter the skin through puncture wounds.
The spores germinate into vegetative cells that produces the
toxin
o Symptoms appear 7 days after injury, but may range from
3-21 days

o Manifestations include muscular rigidity, usually in the jaw,

neck, and lumbar region
o Difficulty in swallowing, rigidity in the abdomen, chest, back,
and limbs, may also occur

o Treatment requires injection of antitoxin, muscle relaxants

o Vaccine: diptheria-tetanus-acellular pertussis (DTaP) vaccine

Clostridium difficile

o Common cause of antibiotic associated diarrhea and

pseudomembranous colitis (bloody diarrhea with necrosis of
colonic mucosa)
o Culture:
 BAP – “horse
stable” odor;
non-hemolytic
 Cycloserine-
cefoxitin-
fructose agar
(CCFA) –
colonies exhibit a
yellow color and
ground glass
appearance

o Microscopy: chains up to 6 cells that are aligned from end to

end with oval, subterminal spores

Gastrointestinal disease

o Following antimicrobial therapy, many bowel flora other than

C. difficile are killed, thus allowing C. difficile to multiply with
less competition and produce two toxins: toxin A
(enterotoxin) and toxin B (cytotoxin)

o Bloody diarrhea with associated necrosis of colonic mucosa is

seen in patient with pseudomembranous colitis

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