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Lec 6 Family Bacillaceae د. انسام محمد
Gram positive spore forming bacilli
Family Bacillaceae
Genus Bacillus
More than 50 species of bacillus are widely distributed in nature. The genus Bacillus
includes large aerobic, catalase positive, gram-positive rods that form heat resistant spore
and the rods are arranged in chains.
On gram staining the spores don’t stain and appear as empty space and doesn’t
distend the vegetative cell. The spores are stained using special spore stain and they are
resistant to environmental changes, withstand dry heat and certain chemical
disinfectants for moderate periods, and persist for years in dry earth.
The spores constitutes the commonest contaminant in bacteriological culture
media. The spores formed during conditions of environmental stress as nutrients
deprivation, temp extremes and drying.
Bacillus subtilis saprophytic organisms prevalent in soil, water, and air.
B.cereus can grow in foods and cause food poisoning by producing a toxin. It may
occasionally produce disease in immune-compromised humans.
B anthracis, which causes anthrax, is the principal pathogen of the genus.
Bacillus anthracis
Morphology
B. anthracis is one of the largest of pathogenic bacteria. 3–8 by 1–1.3 μm and is gram-
positive, non-motile (unlike most other members of this genus), straight, sporing
bacillus. It is rectangular in shape and arranged in filamentous chains in culture, end to
end in long chains ‘bamboo stick’ appearance.
The spore is oval, central in position and not swelling the mother cell. Spores are
formed in culture, in the soil, but never in the blood or tissues of living animals.
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Cultural Characteristics
It is aerobe and facultative anaerobe. Temperature range for growth is 15–45°C
1. Nutrient agar: colonies are irregularly round, 2–3 mm in diameter, raised, dull,
opaque, grayish white, with a frosted glass appearance. The edge of the colony
resembles locks of matted hair under the low power microscope. This is called the
‘Medusa head appearance’.
2. Blood agar: Colonies on horse or sheep blood agar are non-hemolytic.
3. In a gelatin stab, there is growth down the stab line with lateral spikes, longer near
the surface, giving an ‘inverted fir tree’ appearance.
4. Selective medium: A selective medium (PLET medium), has been devised to isolate
B. anthracis from mixtures containing other spore-bearing bacilli.
Resistance
1- With moist heat, the vegetative bacilli are killed at 60°C in 30 minutes and the
spores at 100°C in 10 minutes.
2- With dry heat the spores are killed at 150°C in 60 minutes.
3- The spores are also killed by 4% formaldehyde or 4% potassium permanganate
in a few minutes.
4- The bacilli are sensitive to benzylpenicillin, streptomycin, tetracyclines,
chloramphenicol, ciprofloxacin, the cephalosporins and sulfonamides.
Virulence Factors
The pathogenesis of B. anthracis depends on two important virulence factors:
1. Capsule: consisting of D-glutamic acid which interferes with phagocytosis. B anthracis
isolates that do not produce a capsule are not virulent.
2. Anthrax toxin: Anthrax toxin consists of three proteins called protective antigen
(PA), edema factor (EF), and lethal factor (LF), each of which individually is nontoxic
but together act synergistically to produce damaging effects. EF with PA, it forms a toxin
known as edema toxin responsible for cell and tissue edema. LF plus PA form lethal
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toxin, which is a major virulence factor and cause of death in infected animals and
humans.
Clinical infections
Anthrax is a zoonosis, Cattle, sheep, goats and other herbivores are naturally
infected. The disease not spread from animal to animal, only through ingestion of food
contaminated with the spores. Anthrax is endemic in developing countries. Humans
become infected incidentally by contact with infected animals or their products.
In humans, the infection is usually accidental or occupational in four forms:
1. Cutaneous Anthrax
It begins 2–5 days after infection as a small papule that develops within a few days into
a vesicle filled with dark bluish black fluid. Rupture of the vesicle reveals a black eschar
at the base, with a very prominent inflammatory ring of reaction around the eschar.
(The name anthrax, which means coal, comes from the black color of the eschar).
This is sometimes referred to as a malignant pustule. The lesion is classically found on
the hands, forearms, or head and is painless. Cutaneous anthrax generally resolves
spontaneously, but 10–20% of untreated patients may develop fatal septicemia or
meningitis.
2. Pulmonary Anthrax
Pulmonary anthrax, known as ‘wool-sorter’s disease, it used to be common in
workers in wool factories, due to inhalation of dust from infected wool.. This is a
hemorrhagic pneumonia with a high fatality rate. Hemorrhagic meningitis may occur as
a complication. The spores from the dust of wool are inhaled; phagocytosed in the
lungs; and transported by the lymphatic drainage to the mediastinal lymph nodes,
where germination occurs. This is followed by toxin production and the development of
hemorrhagic mediastinitis and sepsis, which are usually rapidly fatal.
In the U.S. bioterrorism inhalation cases of 2001, the pathogenesis was the same as in
inhalation anthrax from animal products. It resulted in 22 cases of anthrax—11
inhalation and 11 cutaneous. Five of the patients with inhalation anthrax died. All of the
other patients survived.
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3. Intestinal Anthrax
Intestinal anthrax is rare and occurs mainly in primitive communities who eat the
carcasses of animals dying of anthrax. An individual may suffer after a day or so from
hemorrhagic diarrhea, and dies rapidly from septicemia.
4. Injection anthrax, has caused outbreaks among persons who inject heroin that has
been contaminated with spores. Characterized by extensive, painless, subcutaneous
edema and the notable absence of the eschar characteristic of cutaneous anthrax.
Patients may progress to hemodynamic instability due to septicemia
Laboratory Diagnosis of Human Anthrax
1. Specimens: Material from a malignant pustule, sputum from pulmonary anthrax,
gastric aspirates, feces or food in intestinal anthrax and in the blood in the septicemic
stage of all forms of the infection. Specimens should be taken before antibiotic therapy
has been instituted.
2. Microscopy: Prepare smears of each specimen and stain with Gram’s Method which
show typical large gram-positive bacilli.
Direct fluorescent antibody test (DFA) for capsule and for polysaccharide (cell wall)
antigen confirms the identification.
3. Culture: Culture the exudate on nutrient agar, blood agar and PLET medium.
Incubate at 37°C for 18 hours. Examine plates for the medusa-head colonies
characteristic of B. anthracis, non-hemolytic on the blood agar plate. Prepare a stained
smear and look for chains of large gram-positive bacilli some of which have central,
oval, non-bulging spores.
4. Confirmatory tests
i. Biochemical and physiological reactions: Demonstration of non-motility, gelatin
liquefaction, inverted fir tree appearance in a gelatin stab, will generally identify B.
anthracis completely.
Toxin production can be demonstrated by immunological or gene probe methods in
reference laboratories.
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5. Serological diagnosis: Serological diagnosis by enzyme-linked immunosorbent assay
(ELlSA) is seldom used diagnostically.
6. Polymerase Chain Reaction (PCR)
A sensitive and specific PCR technique has been developed for the detection of anthrax
contamination of animal and agricultural products.
Treatment
Ciprofloxacin is the drug of choice. Penicillin, doxycycline, erythromycin, or
chloramphenicol can be used (if susceptible).
Immunization
Live-attenuated bacilli were first used by Louis Pasteur in May 1881. Pasteur’s vaccine
was the anthrax bacillus attenuated by growth at 42–43°C.
Alum-precipitated toxoid prepared from the protective antigen has been shown to be
a safe and effective vaccine for human use. It has been used in persons occupationally
exposed to anthrax infection.
BACILLUS CEREUS
B cereus produces toxins that cause disease that is more of intoxication than a food-
borne infection.
1- Food poisoning caused by B cereus has two distinct forms
a. The emetic type, which is associated with fried rice, milk, and pasta. It is manifested
by nausea, vomiting, abdominal cramps, and occasionally diarrhea and is self-
limiting, with recovery occurring within 24 hours. It begins 1–5 hours after ingestion
of a plasmid-encoded preformed emetic toxin in the contaminated food products.
When rice are cooked and allowed to cool slowly, the B cereus spores germinate,
and the vegetative cells produce the toxin during log-phase growth or during
sporulation.
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b. The diarrheal type, which is associated with meat dishes and sauces. It has an
incubation period of 1–24 hours and is manifested by profuse diarrhea with
abdominal pain and cramps; fever and vomiting are uncommon.
2- Eye infections, such as severe keratitis and endophthalmitis. Typically, the
organisms are introduced into the eye by foreign bodies associated with trauma
but infections can also occur after surgery.
3- Localized infections, such as wound infections, and with systemic infections,
including endocarditis, catheter-associated bacteremia, central nervous system
infections, osteomyelitis, and pneumonia; the presence of a medical device or
intravenous drug use predisposes to these infections.
B cereus is resistant to a variety of antimicrobial agents, including penicillins and
cephalosporins. Serious non–food borne infections should be treated with vancomycin or
clindamycin with or without an aminoglycoside. Ciprofloxacin has been useful for the
treatment of wound infections.
References
1- Koneman's color atlas and textbook of diagnostic microbiology 7
th
edition, 2017.
2- Bailey and Scott's Diagnostic Microbiology 14
th
edition, 2017.
3- Jawetz, Melnick and Adelberg's medical microbiology 26
th
edition, 2013.