Gram Positive Bacilli

Differentiation of Major Gram Positive Rod Genera

I. Based on Gram stain and formation of spores, and catalase reaction
a. Spore forming, catalase positive (aerobic) : Bacillus
b. Regularly shape, non sporing
i. Catalase positive : Listeria monocytogenes
ii. Catalase negative : Erysipelothrix ruthsiopathiae, Lactobacillus spp, Gardnerella
vaginalis
c. Irregularly shaped, non-sporing, catalase positive : Corynebacterium spp
d. Branching : Nocardia spp, Actinomyces sp, and Streptomyces sp
Spore-Forming Aerobic or Facultative Anaerobic Bacilli – CATALASE POSITIVE
I. Bacillus species
a. Epidemiology : widely distributed in nature
b. Virulence
i. Production of endospores
ii. Spores produced when bacteria is stressed
iii. Some species produces capsules and exotoxins
c. Clinical Significance
i. Environmental contaminants or normal flora
ii. B. anthracis causes anthrax
iii. Opportunistic infections – serious infections on immunocompromised patients
d. Laboratory Identification
i. Gram Stain
1. Large aerobic or facultative anaerobic gram positive rods that form
endospores
2. May or may not exhibit exospores
3. Spores produced when bacteria in stress such as drying conditions, and
unfavourable temperatures
4. Appear as clear areas with bacterial cell on gram stain
5. Spores does not distened the cell wall
6. Aerobic production of spores only
7. Organism can be heated shock to form spores ; heat suspension in 56C water
bath
8. May stain gram variable or negative
9. Perform 3% KOH test to establish gram reaction needed
 Place a drop of 3% KOH on slide, with a loop emulsify KOH on slide
gently raised the loop up approx. 0.5-1cm
 Viscous string : bacterial DNA (follows loops : gram neg
 No viscous ring : gram positive
ii. Colonial morphology
1. SBA or CHOC with 24hours at 35C in ambient air or 5% CO2
2. Colonies are usually large, flat with frequent hemolysus
iii. Presumptive identification – to rule out B. anthracis
1. B-hemolytic on SBA : B. anthracis is non-hemolytic
2. Motile : B. anthracis is non-motile
II. Bacillus anthracis
a. Epidemiology : Widely distributed in nature
i. Cause disease in human and animals
1. Animals infected by feeding plants contaminated with spores
2. Human primarily infected as result of contact with animals or animal products
 Animal hides, fibers, or other animal products
 Inhalation or traumatic intorduction
b. Virulence factors
i. Antiphagocytic capsule
ii. Exotoxins that mediate cell and tissue destruction
1. Edema and lethal toxin
2. Protective factor
c. Clinical Significance
i. One of the most microorganisms for humans : bioterrorism agent
ii. Causative agent for anthrax
1. Cutaneous anthrax
 Site of spore penetration
 Ulceration to form black eschar
 Lead to fatal toxaemia (approx. 20% mortality)
 2. Pulmunary anthrax (Woolsorter’s disease)
 Inhalation of spores
 Respiratory distress, chest edema, cyanosis, death
 10% fatal if not treated early
3. Gastrointestinal anthrax
 Ingestion of spores
 Most patient dye from toxaemia and overwhelming sepsis
d. Laboratory Identification
i. Gram Stain
1. Large, square ended, gram positive rods in chains or single
 Subterminal spore
ii. Colony Morphology
1. Non-hemolytic, large, gray flat colonies with irregular margins
2. Filamentous projections – Medusa head on SBA
3. Bicarbonate agar in 5% CO2 will induce capsule formation-mucoid
iii. Preliminary Identification
1. Non-hemolytic, non-motile colonies should be sent to reference laboratory
state health laboratory for confirmatory identification (cases reported to state
and CDC)
2. Sensitive to penicillin
e. Treatment/Antibiotic therapy/Prevention
i. DOC : penicillin – supportive therapy may be needed
ii. Animal vaccine : responsible for reducing incidence
iii. Human vaccine available (military and health care workers0
III. Bacillus cereus
a. Epidemiology
i. Widely distributed in nature, human GIT
b. Virulence Factors
i. Toxins (emetic or enterotoxin)
c. Clinical Significance
i. Food poisoning : food contaminated with organism or toxin formed by organism
1. Diarrheal type :
 Abdominal pain and watery diarrhea due to enterotoxin
 Associated with fried or boiled rice
 Symptoms usually 1-6 hours after ingestion
 Recover 6-24 hours after onset
2. Emetic type
 Vomiting caused by emetic toxin
 Poultry, cooked meats, soups and desserts
 Symptom usually shows 10-12 hours after ingestion
 Recover 12 hours after onset
ii. Serious infection in immunocompromised host
1. Traumatic eye wounds
2. Endocarditis
3. Bacteremia a
4. Wounds
d. Laboratory Identification
i. B. cereus is normal stool flora, to diagnose food poisoning must culture suspected
food not stool
ii. Gram stain : large gram positive rods with spores, can stain gram variable or gram
negative
iii. Colony morphology : beta haemolytic, large, feathery, spreading on SBA
iv. Preliminary identification
1. Beta haemolytic
2. Motile
3. Penicillin resistant
Non-Spore-Forming Bacilli, CATALASE POSITIVE
I. Listeria species : Listeria monocytogenes
a. Pathogenesis
i. Bacteremia and meningitis (immune-compromised host)
ii. Pregnant women may pass the infection to the fetus causing systemic infection and
stillbirth
iii. Ingestion of contaminated food : meat and dairy products
b. Isolation and Identification
i. BAP : 24-48 hours : beta-hemolytic (small zone) ; small grayish, translucent, 30-35C
in ambient air or 5% CO2 [Facultative]
ii. Gram stain
1. Small gram positive rods (almost coccal), may be in pairs or in short chains
2. Non-sporulating
iii. Identification
1. Catalase : positive
2. Tumbling motility : on wet preparation
3. Umbrella-shaped motility : semi solid media at RT
4. Esculin hydrolysis : positive
5. Sodium hippurate hydrolysis positive
6. Ferments glucose
7. Cold enrichment (will grow at 4C)

II. Corynebacterium species- diphtheroids

a. General Characteristics
i. Widely distributed in Nature
ii. Many species are normal flora of skin and mucous membranes
iii. Most species are non-pathogenic (referred collectively as diphtheroids)
b. Morphology
i. Gram Stain :
1. Club shaped and beaded with irregularly staining granules, pleomorphic
(many size and shape), palisading (Chinese letters) gram positive rods
2. Non-sporing
c. Characteristics
i. Catalase positive
ii. Albert’s stain (LAMB stain) – Babst Ernst granules or metachromatic granules are
seen in the organism cells (specific for Corynebacterium sp)
iii. Glucose fermentation/oxidation : variable
iv. Sucrose fermentation/oxidation : variable
v. Urease : variable
vi. Nitrate reduction : variable
III. Corynebacterium diphtheriae – diphtheria
a. Diphtheria
i. Disease of the respiratory tract
ii. Pseudomembrane : should be cultured, if not present then culture nose, throat or
wound
iii. Toxigenic vs non-toxigenic (exotoxin-toxin)
1. Toxin producing : infected with beta bacteriophages
2. Toxin blocks protein synthesis
b. Pathogenesis
i. Found primarily on the epithelial cells of the respiratory tract of persons with disease
or carriers
ii. Infection occurs by droplets or contact to susceptible (no or low antitoxin) individuals
iii. During infection that causes organism localize in upper respiratory tract and
produces exotoxin that causes necrosis forming a grayish pseudo-membrane (WBC
and organism)
iv. Toxin is absorbed into the blood and affects the myocardium and peripheral nervous
system. Death is usually due to congestive heart failure
c. Treatment and Prevention
i. Treatment : antitoxin is given in the form of toxoid
ii. Prevention : DPT immunization
d. Isolation and Identification
i. BAP : 24-48 hours at 35C in ambient or 5% CO2 : small, gray, translucent colonies to
medium, white opaque colonies
ii. Gram Stain
 1. Irregular staining, pleomorphic gram positive rods
iii. Identification
1. Loeffler’s media : used for isolation for Corynebacterium sps, enhances the
granule formation as seen on Albert’s stain and characteristic cellular
morphology of C. diphtheriae
2. Tellurite Media (modified Tinsdale agar) :
a. tellurite is reduced to metallic tellurium by Corynebacterium spp.
causing colonies to appear grayish black.
b. C. diphtheriae can be differentiated from other diphtheroids by having
a brown halo around the colony
3. Non-lipophilic
4. Glucose fermentative
5. Sucrose negative
6. Urea negative
7. Nitrate Reduction Variable
IV. Corynebacterium jekeium
a. Disease State
i. Immuno-compromised patients : septicaemia, meningitis, pulmonary disease
b. Isolation and Identification
i. BAP : 48-72 hours at 35 C in ambient air or 5% CO2 – small gray to white colony (non-
hemolytic)
ii. Gram Stain
1. Pleomorphic ; occasionally, club shaped gram positive rods arranged in V
forms or palisades
iii. Identification L
1. Lipophilic(growth is enhanced with lipid added to media such as Tween 80)
2. Glucose oxidative
3. Sucrose negative
4. Urea negative
5. Nitrate reduction negative
iv. Susceptibility testing
1. Resistance to multiple antibiotics used to treat gram positive infections
2. To date al isolates have been susceptible to vancomycin
Non-Spore-Forming Bacilli, CATALASE NEGATIVE
I. Gardnerella Species
a. Gardnerella vaginalis
b. Morphology and Characteristics
i. Colonial Morphology
1. Does not grow on SBA
2. Growth on Human Blood Agar (V, HBT) shows beta hemolysis
ii. Gram Stain
1. Pleomorphic, gram variable rod
2. Non-sporulating
3. Specimen gram stain : CLUE CELLS : epithelial cells covered with tiny bacilli
especially around the edge
iii. Identification
1. CATALASE negative
2. Sodium hippurate : usually positive
3. SPS : sensitive
c. Disease state : Bacterial Vaginosis
i. A polymicrobic infection with Mobilincus and Bacteroides
ii. Diagnosis : presence of homogenous, gray discharge, clue cells seen on Gram stain
or wet mount, amine or fishy odor when 1 drop 10% KOH added to discharge on slide
iii. Culture is not recommended for diagnosis
II. Erysipelothrix species
a. Erysipelothrix rhusiopathiae
b. Morphology and Characteristics
i. Colonial Morphology
1. Microaerophilic
2. Non-hemolytic or beta hemolytic
ii. Gram Stain
1. Both short gram positive rods and long filamentous rods corresponding to two
colony types
2. Non-spurolating
iii. Identification
1. CATALASE negative
2. Non-motile
3. Test tube brush growth pattern in semisolid motility tube after 48 hours
incubation at room temperature
4. H2S on TSI or KIA – positive (the only GPR that is H2S positive)
5. Sucrose non fermenting
c. Disease States : Erysipelas
i. Zoonotic
ii. In swine, it produces an important economic disease called swine erysipelas that is
generally fatal
iii. Man becomes infected by coming in direct contact with an infected animal
iv. Organisms enters abraded skin (often finger or hand)
v. A skin disease that is characterized by intense pain and is usually self-limited
vi. Rare cases becomes serious, disseminating to septicaemia with arthritis or
endocarditis
vii. There is no permanent immunity and relapses are common
III. Lactobacilli
a. Morphology and Characteristics
i. Colonial Morphology
1. Microaerophilic : incubate in 5-10%
2. May show alpha hemolysis in blood agar
ii. Gram Stain
1. Long slender gram positive rods in chains, or short coccobacilli
2. Non-spurolating
iii. Identification
1. CATALASE negative
2. Sucrose fermenting
3. Resistant to vancomycin [aid in identification]
b. Normal Flora : mouth, gastrointestinal tract, female genital tract
c. Disease state :
i. Rare pathogenic : implicated in rare cases of endocarditis and meningitis
Branching Norcardioform Bacilli
I. Nocardia Species
a. Norcardia asteroids, N. braziliensis, N. caviae
b. Morphology and Characteristics
i. Colonial Morphology
1. Aerobic growth appears in 3-30 days
2. Waxy, bumpy or velvety rugose forms, yellow to Orange color
3. Will grow on SBA, mycology media and LJ media
ii. Gram Stain
1. Pleomorphic, branching, fine, delicate filaments with fragmentation
2. Gram positive rods that are often beaded in appearance
iii. Identification : Partially acid fast positive
c. Habitat : soil and water
d. Disease State
i. Mycetoma (Actinomycetoma) – a chronic, localized, painless, subcutaneous infection
1. Tissue swelling
2. Draining sinus tracts
3. Presence of Granules
ii. Lymphocutaneous infections
iii. Skin abscesses or cellulitis
iv. Immunocompromised patients : pulmonary and disseminated infections
II. Streptomyces Species
a. Streptomyces somaliensis, S. anulatus, S. paraguayensis
b. Morphology and Characteristics
i. Colonial Morphology
1. Aerobic growth appears in 3-30 days
2. Waxy, bumpy or velvety rugose forms, yellow to Orange color
3. Will grow on SBA, mycology media and LJ media
ii. Gram Positive
1. Gram positive rods with extensive branching, chains and spores
2. Does not fragment easily
iii. Identification : acid fast negative
c. Habitat : Soil and Decaying vegetation
d. Disease State : Mycetoma (Actinomycetoma) – a chronic, localized, painless, subcutaneous
infection