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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY

DIAGNOSTIC MICROBIOLOGY | Non-Branching, Spore-forming Catalase-Positive, Gram-Positive


Bacilli

GENERA AND SPECIES TO BE CONSIDERED

֍ Bacillus Cereus Group


 Bacillus anthracis
 Bacillus cereus (type species)
 Bacillus mycoides
 Bacillus megaterium
 Bacillus thuringiensis
 Bacillus cytotoxicus
֍ Bacillus Circulans Group
 Bacillus circulans (type species)
 Bacillus firmus
 Bacillus lentus
 Bacillus coagulans
֍ Bacillus Subtilis Group
 Bacillus licheniformis
 Bacillus amyloliquefaciens
 Bacillus subtilis (type species)
 Bacillus pumilus
֍ Others
 Brevibacillus spp.
 Paenibacillus spp.

GENERAL CHARACTERISTICS

1. metabolically diverse, and some species are thermophiles that grow best at 55° C or higher
2. Most are saprophytes
3. aerobic or facultative anaerobic
4. Motile except B. anthracis, and B. mycoides
5. Large bacilli that resemble bamboo-fishing rods

CLINICALLY SIGNIFICANT ISOLATES

֍ Bacillus anthracis / Anthrax Bacilli


 Bioterrorism Agent
 Features
 Comet tail / medusa head colonies
 Ground glass colonies
 Non-motile, Non-hemolytic
 ferments glucose, but it fails to ferment mannitol, arabinose, or xylose
 lecithinase producer
 grows in high-salt (7% sodium chloride) and low pH (<6) conditions
 generally susceptible to penicillin (10 U/mL)
 Virulence
 glutamic acid capsule
 Plasmid-borne anthrax toxins containing the protective antigen and a funtional enzyme
 Lethal Toxin : Lethal Factor
 Edema Toxin : Edema Factor
 Diseases and Epidemiology
 the most highly virulent species and is the causative agent of anthrax
 found in soil (reservoir) and is usually contracted by herbivores
 Four forms of disease
 Cutaneous anthrax
⸋ accounts for most human infections
⸋ associated with contact with infected animal products.
⸋ Infection results from close contact and inoculation of endospores
through a break in the skin.
⸋ After inoculation and an incubation period of approximately 2 to 6 days
in most cases, a small papule appears that progresses to a ring of
vesicles, and then develop into an ulceration.
⸋ The typical presentation of the ulceration is a black, necrotic lesion
known as an eschar (called malignant pustule)
⸋ Fatalities may occur when the lesions form on the face or neck and
cause an obstructed airway after edema or progression to systemic
disease
 Ingestion anthrax
⸋ results from ingestion of endospores and presents in two forms:
⸋ oral or oropharyngeal,with the lesion appearing in the buccal cavity or
on the tongue, tonsils, or pharyngeal mucosa,

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | Non-Branching, Spore-forming Catalase-Positive, Gram-Positive
Bacilli


gastrointestinal anthrax, with the lesions developing typically in the
mucosa of the terminal ileum or cecum.
 Inhalation anthrax
⸋ Pulmonary anthrax
⸋ Inhalation of endospores which are ingested by macrophages in the
lungs and transported to the lymph nodes, leading to the development
of systemic infection.
⸋ The disease appears flulike and includes symptoms such as fever,
chills, fatigue, a nonproductive cough, and nausea or vomiting that then
progresses to respiratory distress, edema, cyanosis, shock, and death.
⸋ Patients typically demonstrate abnormal chest x-rays with pleural
effusion, infiltrates, and mediastinal widening.
⸋ Woolsorters’ disease and ragpickers’ disease are terms used to describe
respiratory infections that result from exposure to endospores during
the handling of animal hides, hair, fibers, and other animal products.
 Injectional Anthrax
 Patients can develop meningitis within 6 days after exposure.
֍ Bacillus cerus group excluding B. anthracis

Crystalforming : B. thuringiensis a probiotic : B. toyonensis

cold-tolerant : B. weihenstephanensis morphologic variants : B. mycoides and B.


pseudomycoides
heat-tolerant : B. cytotoxicus;

 Features
 Motile
 Beta-hemolytic
 Frosted Glass appearance on SBA
 Virulence
 hemolysin BL (HBL), nonhemolytic enterotoxin (Nhe), and cytotoxin K (CytK; also
referred to as hemolysin IV). The three toxins are believed to act synergistically, with
Nhe responsible for the major symptoms in the diarrheal presentation of the infection.
 The emetic form of illness is associated with a heat-stable, proteolysis, and acid-resistant
toxin, cereulide, which is produced in food.
 The cereulide toxin is encoded on a plasmid-borne gene cluster and has been identified in
a subset of B. cereus and B. thuringiensis isolates.

 Diseases and Epidemiology


 found within the soil andwidely distributed in nature
 opportunistic pathogens and often associated with foodborne illness
 food poisoning is associated with the ingestion of a wide variety of foods including
meats, vegetables, desserts, sauces, and milk. A higher incidence is seen after the
ingestion of rice dishes.
 The organisms may be associated with localized skin infections or systemic conditions
such as bacteremia, endocarditis, and septicemia. Additional sites of infection include the
urinary or respiratory tract.
 Health care–associated infections have also been identified and linked to contaminated
material such as gowns, gloves, linens, and dressings, as well as medical devices
including catheters, ventilators, and bronchoscopy equipments
 Infections with B. cereus group organisms have also been linked to contaminated drugs.

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | Non-Branching, Spore-forming Catalase-Positive, Gram-Positive
Bacilli

 eye infections are the most common type of nongastrointestinal infection caused by
B.cereus organism
 Bacillus thuringiensis
 Insect pathogen
 Harbors the cerulide enterotoxin
 Toxins have been commercialized for the control of insects that cause agricultural
damage, such as moths, beetles, flies, and parasitic worms (nematodes)
 Occupational exposure to insecticides and pesticides containing the organism has resulted
in the identification of the organism in feces without the presence of gastrointestinal
symptoms. Additional rare cases of wound, burn, pulmonary, and ocular infections have
been attributed to B. thuringiensis.

LABORATORY DIAGNOSIS AND IDENTIFICATION

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | Non-Branching, Spore-forming Catalase-Positive, Gram-Positive
Bacilli

SUMMARY OF IDENTIFICATION

֍ All Bacillus and related genera grow well on 5% sheep blood agar, chocolate agar, routine blood culture
media, and nutrient broths.
 Isolates susceptible to nalidixic acid will not grow on Columbia agar with nalidixic acid and
colistin (CNA), a selective and differential medium for gram-positive organisms.

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | Non-Branching, Spore-forming Catalase-Positive, Gram-Positive
Bacilli

 Phenylethyl alcohol agar (PEA), an additional selective agar for gram-positive organisms, is
useful for the removal of contaminating organisms and the isolation of Bacillus spp.
 Polymyxin-lysozyme-EDTA-thallous acetate (PLET) can be used for selection and isolation
from contaminated specimens.
 Beaten Egg White / Teancious Consistency of Colonies
 Bicarbonate agar is used to induce B. anthracis capsule formation, providing a means for
presumptive morphologic identification.
 Requires CO2 incubation
 B. cereus media referred to as mannitol, egg yolk, and polymyxin B agar (MEYP or MYP);
polymyxin B, egg yolk, mannitol, bromothymol blue (PEMBA); and B. cereus medium (BCM)
have been developed for the specific isolation and identification of the organism.
֍ Most species will produce detectable growth within 24 hours after incubation at 35°C, in ambient air, or in
5% carbon dioxide (CO2).
 Colonies appear as creamy white, domed, circular colonies.
֍ Heat shock treatment can be used for the growth and enhancement of endospores from clinical specimens.
֍ Sporulation is inhibited by high concentrations of CO2
 Spores may be induced by growth in in triple sugar iron (TSI), urea, or nutrient agar containing 5
mg/L manganese sulfate.
 Special staining is required to visualize endospores
 Schaeffer–Fulton : Malachite Green and Safranin
 endospores stain green
 vegetative cells will appear pink from the secondary stain, safranin
֍ Direct examination of blood smears may also reveal encapsulated rods or “ghost” capsules after antibiotic
treatment when stained using polychrome methylene blue (M’Fadyean).
 Capsule production by B. anthracis can be detected by India ink staining on blood or CSF
specimens or on cells isolated in media supplemented with sodium bicarbonate, although this
technique is not routinely performed by clinical laboratories
֍ B. anthracis is catalase positive and grows aerobically or anaerobically.
 Although B. anthracis ferments glucose, it fails to ferment mannitol, arabinose, or xylose.
 B. anthracis produces lecithinase; an opaque zone can be seen around colonies growing on egg-
yolk agar.
 This species grows in high-salt (7% sodium chloride) and low pH (<6) conditions.
 In contrast to B. cereus, B. anthracis is generally susceptible to penicillin (10 U/mL).
֍ B. anthracis is nonmotile, distinguishing it from most other members of the genus
 B. mycoides is also nonmotile.
 Motility can be tested by either wet mount preparation or inoculation into motility test medium.
֍ On SBA, colonies of B. anthracis are nonhemolytic, large (2 to 5 mm), gray, and flat with an irregular
margin because of outgrowths of long, filamentous projections of bacteria that can be seen with a dissecting
microscope.
 The term Medusa head has been used to describe the colony morphology of B. anthracis.
 Colonies have a tenacious consistency, holding tightly to the agar surface, and when the edges are
lifted with a loop, they stand upright without support.
 described as having the appearance or characteristic of beaten egg whites
֍ The current CDC level A testing protocol for the presumptive identification of anthrax recommends using
PEA agar for stools suspected to contain B. anthracis, in addition to SBA and other commonly used media.
֍ Cutaneous anthrax specimens should be collected from underneath the eschar.
 Two specimens of the vesicular fluid should be collected from underneath the lesion by rotating
the swabs beneath the eschar.
 For histochemical testing, the physician may collect a punch biopsy that should be placed in 10%
formalin.
֍ Inhalation anthrax specimens should include blood cultures, pleural fluid, and a serum specimen for
serology.
 physician may collect a biopsy of bronchial or pleural tissue.
֍ Specimens required for gastrointestinal anthrax include blood cultures, ascites fluid, and material from any
lesions as well as serum for serologic testing.
֍ The preferred collection of specimens from patients suspected of infection with B. anthracis should be
accumulated before antibiotic therapy.
 Specimens should also be collected from the potential source of infection, such as the animal
carcass.
֍ Despite the publicity associated with B. anthracis as a potential agent of biologic warfare, the organism is
not highly contagious.
 disinfection with formaldehyde, glutaraldehyde, or hydrogen peroxide and peracetic acid should
be performed before the disposal of specimens suspected of containing a large number of spores.
֍ B. anthracis is classified by the Department of Health and Human Services/Centers for Disease Control and
Prevention (CDC) and the U.S Department of Agriculture/Animal and Plant Health Inspection Service
(APHIS) as a select agent.

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UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY
DIAGNOSTIC MICROBIOLOGY | Non-Branching, Spore-forming Catalase-Positive, Gram-Positive
Bacilli


Any laboratory in possession of the organism must register with one of these agencies and notify
the organization within 7 days upon identification of the organism.
 If the organism is identified in an unregistered laboratory, the isolate must be shipped, using the
request to transfer select agents and toxins approval from CDC or APHIS, to a registered
laboratory for proper disposal.
֍ Culture of suspected food from a food poisoning incident may be done to isolate and quantify B. cereus.
 If more than 105 B. cereus cells per gram of food are present and other pathogens are absent, food
poisoning by this organism is confirmed.
֍ A β-hemolytic frosted glass–appearing colony containing spore-forming, gram-positive bacilli that are
motile, able to ferment salicin, and lecithinase positive is likely B. cereus.
 B. cereus be grown aerobically at 37° C on SBA.

ANTIMICROBIAL SUSCEPTIBILITY TESTING AND THERAPY

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