Aerobic

Gram-Positive
Bacilli
Rochelle D. Darlucio, RMT, MPH
College of Medical Laboratory Science
Our Lady of Fatima University
 Non–Spore-Forming, Nonbranching,
Catalase-Positive Bacilli

• Corynebacterium
• Rothia
• Listeria
 Corynebacterium

• General Characteristics

• slightly curved, gram-positive rods with nonparallel

sides and slightly wider ends, producing the described
“________________” or _____________.
• Diphtheroid, meaning “______________________”
• Can be divided into nonlipophilic and lipophilic species.
 Corynebacterium
• Corynebacterium diphtheriae

• VIRULENCE FACTORS:
• Diphtheria toxin
• major virulence factor
• This toxin is produced by strains of C. diphtheriae infected with a
lysogenic β-phage, which carries the gene tox for diphtheria toxin.
• protein of 62,000 daltons (Da).
• Composed of 2 fragments:
• _________________
• _________________
 Corynebacterium

Corynebacterium diphtheriae
• Clinical Infections
• Two different forms of disease in humans:
• _____________________: sudden onset with exudative
pharyngitis, sore throat, low-grade fever, and malaise; a thick
pseudomembrane develops over the pharynx; in critically ill
patients, cardiac and neurologic complications are most
significant
• _____________________: a papule can develop on the skin,
which progresses to a nonhealing ulcer; systemic signs can
develop.
 Corynebacterium
• Laboratory Diagnosis
• MICROSCOPY
• highly pleomorphic gram-positive bacillus
• appears in palisades (cells lie in parallel
rows) or as individual cells lying at sharp
angles to another in “V” and “L”
formations
• club-shaped swellings and beaded forms
are common
• often stain irregularly, especially when
stained with methylene blue,
• The metachromatic areas of the cell, which
stain more intensely than other parts, are
called _____________________
• Culture Characteristics
• facultative anaerobe
• It grows best under aerobic conditions and
has an optimal growth temperature of 37° C,
although multiplication occurs within the
range of 15° to 40° C.
• grows on nutrient agar, better growth is
usually obtained on a medium containing
blood or serum, such as ________________
or Pai agars
• have a very small zone of β-hemolysis.
• Cystine-tellurite blood agar (CTBA), a
modification of Tinsdale medium, contains
sheep red blood cells, bovine serum, cystine,
and potassium tellurite.
• When grown on CTBA, corynebacteria form
black or brownish colonies from the
reduction of tellurite.
 Corynebacterium
• Laboratory Diagnosis
• IDENTIFICATION
• catalase positive
• Nonmotile
• Brown halo on CTBA
• _______________________
• _______________________
• _______________________
• C. diphtheriae
• lacks urease production
• ferments glucose and
maltose
• producing acid, but not gas
• reduces nitrate to nitrite
• TEST FOR TOXIGENITY
• In Vitro –
• ______________– Immunodiffusion
test
(+) Lines of precipitation
• TREATMENT
• Diphtheria is treated by prompt
administration of antitoxin.
• Drug of choice: Penicillin
• Erythromycin- used for penicillin-
sensitive individuals
Corynebacterium
Corynebacterium
 Other Corynebacteria
Corynebacterium amycolatum
• most frequently recovered from
human specimens.
• It is part of the normal skin microbiota
and has often been misidentified by
clinical laboratories as C. striatum, C.
xerosis, and C. minutissimum.
• Flat and dry, have a matte or waxy
appearance, and are nonlipophilic.
• Resistant to a wide range of
antimicrobials, including β-lactams,
fluoroquinolines, macrolides,
clindamycin, and aminoglycosides.
Corynebacterium jeikeium
• named after Johnson and Kaye
• most common cause of
Corynebacterium-associated prosthetic
valve endocarditis in adults.
• also causes septicemia, meningitis,
prosthetic joint infections, and skin
complications, such as rash and
subcutaneous nodules.
• lipophilic and a strict aerobe that is
nonhemolytic, does not produce urease,
and is nitrate reduction negative.
• resistant to a wide range of
antimicrobials, including penicillins,
cephalosporins, macrolides, and
aminoglycosides.
• Drug of choice: vancomycin.
 Other Corynebacteria

• Corynebacterium pseudodiphtheriticum • Corynebacterium pseudotuberculosis

• part of the normal biota of the human • primarily a veterinary pathogen.
nasopharynx
• respiratory tract infection can mimic • causes a granulomatous
lymphadenitis in humans.
respiratory diphtheria
• does not show the characteristic • produces a dermonecrotic toxin that
causes death of various cell types, and
pleomorphic morphology
it can produce diphtheria toxin.
• often appear in palisades. • also produces a brown halo on CTBA.
• grows well on standard laboratory • produces urease and on SBA forms
media, reduces nitrate, and produces
small, yellowish-white colonies
urease
 Other Corynebacteria
• Corynebacterium ulcerans
• Corynebacterium striatum • has been isolated from humans with
• often considered a commensal or skin diphtheria-like illness
contaminant. However, nosocomial • a veterinary pathogen, causing
infections have been reported mastitis in cattle and other domestic
• nonlipophilic and pleomorphic, and it and wild animals.
often produces small, shiny, convex • produces a brown halo around
colonies in about 24 hours. colonies on CTBA.
• shown resistance to penicillins and other • grow well on SBA and produce a
β-lactams, macrolides, and narrow zone of β-hemolysis.
fluoroquinolones but is typically
susceptible to vancomycin.
• does not reduce nitrate,
differentiating it from C. diphtheriae,
and it is urease positive
 Other Corynebacteria

• Corynebacterium urealyticum
• most commonly associated with UTIs.
• lipophilic and is a strict aerobe.
• pinpoint, nonhemolytic, white colonies that have characteristic coryneform
microscopic morphology
• nitrate negative, catalase positive, and urease positive within minutes after
inoculation on a Christensen urea slant.
• The drug of choice for C. urealyticum infections is vancomycin.
 Rothia
• gram-positive cocci that can appear rodlike, belong to the family Micrococcaceae.
• nitrate positive
• Nonmotile
• esculin hydrolysis positive
• urease negative
• Approximately two thirds of the isolates are catalase positive.
• Contains 6 spp. but only 2 are significant
1. _______________________________ - linked to bacteremia, endocarditis, pneumonia
2. ________________________________-member of the normal human oropharyngeal
flora and found in saliva
• Microscopic characteristics:
1. Resembles coryneform bacilli, forming short gram-positive bacilli but also branching
filaments that resemble filaments of facultative actinomycetes
2. When placed in broth, the species produces coccoid cells, a characteristic differentiating it
from actinomycetes
 Listeria monocytogenes
• has been recovered from soil; water; vegetation; and animal products, such as raw
milk, cheese, poultry, and processed meats.
• It also has been isolated from crustaceans, flies, and ticks.
• ___________________________ is recognized as an uncommon but serious
infection primarily of neonates, pregnant women, older adults, and
immunocompromised hosts.

• VIRULENCE FACTORS:
• hemolysin (listeriolysin O)
• Catalase
• superoxide dismutase
• phospholipase C
• surface protein (p60).
 Listeria monocytogenes
• LABORATORY DIAGNOSIS
• MICROSCOPY
• CLINICAL INFECTIONS • gram-positive coccobacillus
• Listeriosis • found singly, in short chains, or
in palisades
• newborns and
immunocompromised
adults- most common
• For healthy individuals,
particularly in pregnant
women, also occurs.
 Listeria monocytogenes
• LABORATORY DIAGNOSIS
• CULTURAL CHARACTERISTICS
• grows well on SBA, chocolate agar, nutrient agars, brain-heart infusion medium and thioglycolate broth.
• prefers a slightly increased carbon dioxide (CO2)
• colonies are small, round, smooth, and translucent, surrounded by a narrow zone of β-hemolysis, which
may be visualized only if the colony is removed.
• Optimal growth temperature for L. monocytogenes is 30° to 35° C, but growth occurs over a wide range
(0.5° to 45° C)
• Because L. monocytogenes grows at 4° C, a technique called cold enrichment may be used to isolate the
organism from polymicrobial clinical specimens.
 Listeria monocytogenes

• IDENTIFICATION
• hippurate hydrolysis positive like S.
agalactiae
• catalase positive
• bile esculin hydrolysis positive
• motile at room temperature
• Wet mount preparations: “__________
____________________” (end-over-
end motility)
• In motility medium: “______________
__________________
• produces a positive CAMP reaction
 Non–Spore-Forming, Nonbranching,
Catalase-Negative Bacilli

• Erysipelothrix rhusiopathiae
• Arcanobacterium and Trueperella
• Gardnerella vaginalis
 Erysipelothrix rhusiopathiae
General Characteristics
CLINICAL INFECTIONS
• three species in the genus Erysipelothrix:
Erysipelothrix rhusiopathiae 1. Erysipeloid
Erysipelothrix tonsillarum 2. septicemia
Erysipelothrix inopinata. 3. generalized, diffuse cutaneous
infection
• gram-positive, catalase-negative
• non–spore-forming
• pleomorphic rod that has a tendency to
form long filaments.
• It is found worldwide and is a commensal or
a pathogen in a wide variety of vertebrates
and invertebrates
• usual route of infection is through cuts or
scratches on skin.
 Erysipelothrix rhusiopathiae
LABORATORY DIAGNOSIS
MICROSCOPY
• thin, rod-shaped, gram positive organism
that can form long filaments
• arranged singly, in short chains, or in a “V”
shape
• decolorizes easily, so it may appear gram
variable
Culture Characteristics
• grows on SBA and chocolate agar
• On SBA, the colonies are usually
nonhemolytic and pinpoint after 24
hours of incubation.
• After 48 hours of incubation, two
distinct colony types are seen:
• A smaller, smooth form is transparent,
glistening, and convex with entire edges
• larger, rough colonies are flatter with a
matte surface, curled structure, and
irregular edges.
• often appear α-hemolytic
Erysipelothrix rhusiopathiae
LABORATORY DIAGNOSIS
IDENTIFICATION
• Catalase-negative
• Nonmotile
• Pleomorphic
• aerobic or facultatively anaerobic
• hydrogen sulfide positive
• Voges-Proskauer test negative
• a gelatin stab culture: “_______________
_________________________________”
pattern at 22° C.
 Arcanobacterium and Trueperella
• A. haemolyticum (formerly Corynebacterium haemolyticum)
• Trueperella (Arcanobacterium) pyogenes
• Trueperella (Arcanobacterium) bernardiae

• Catalase Negative

• A. haemolyticum
• produces small colonies on SBA that demonstrate a narrow zone of β-hemolysis after
24 to 48 hours of incubation
• a black opaque dot is observed on the agar when the colony is scraped away.
• Pitting of the agar beneath the colony has also been reported
• lipase and lecithinase positive
• exhibits a __________________________________________(CAMP inhibition reaction)
 Gardnerella vaginalis
• normal biota in the human urogenital tract.
• short, pleomorphic gram-positive rod or
coccobacillus that often stains gram variable or
gram negative.
CULTURE CHARACTERISTICS
• known for its association with bacterial vaginosis
(BV) in humans • grows best in 5% to 7% CO2 at a
• MICROSCOPY: “________________________” temperature of 35° to 37° C
aids the diagnosis of BV • grows on SBA as pinpoint,
• Amsel’s clinical criteria: nonhemolytic colonies
(1) homogeneous, thin, white discharge that
smoothly coats the vaginal walls
• Medium of choice: ____________
____________________________
(2) clue cells
(3) pH of vaginal fluid greater than 4.5, and
(4)fishy odor of vaginal discharge before or after
addition of 10% potassium hydroxide, the whiff test.
 Non–Spore-Forming, Branching, Aerobic
Actinomycetes

• Nocardia
• Other Actinomycetes
• Actinomadura
• Streptomyces
• Gordonia
• Rhodococcus
• Tropheryma whipplei
 Nocardia
GENERAL CHARACTERISTICS
• aerobic, branched, beaded, gram-
positive bacilli
• partially acid fast
• Virulence Factors
• No virulence factors have been
identified, although virulence has
been correlated with alterations in the
components in the cell wall.
• superoxide dismutase and catalase
• ironchelating compound called
nocobactin.
• Clinical Infections
• Pulmonary infection
• most common manifestation: confluent
bronchopneumonia
• Sputum is thick and purulent
• no sulfur granules develop, and no sinus
tract formation occurs.
• Cutaneous infection
• N. brasiliensis is the most frequent cause
of this form of nocardiosis and
actinomycotic mycetomas
• pus may be pigmented and contain
“________________________________”
• granules often appear yellow or orange
and have a distinct granular appearance
 Nocardia

• Culture Characteristics
• Modified Thayer-Martin agar, may
enhance recovery of Nocardia spp. by
inhibiting the growth of contaminating
organisms.
• grow on nonselective ________________
___________________________________
• a chalky, matte, velvety, or powdery
appearance and may be white, yellow,
LABORATORY DIAGNOSIS pink, orange, peach, tan, or gray
pigmented.
• MICROSCOPY • dry, crumbly appearance similar to
• gram-positive, beaded, branching breadcrumbs
filaments
 Other Actinomycetes
• Actinomadura
• Actinomadura madurae
• Actinomadura pelletieri
• causes mycetomas
• The microscopic morphology and
colony morphology are very similar to
those of Nocardia spp.
• ________________________is
cellobiose and xylose positive,
whereas Nocardia spp. do not
produce acid from these two
carbohydrates.
• Streptomyces
• primarily saprophytes found as soil
inhabitants and resemble other aerobic
actinomycetes with regard to
morphology and the diseases they cause.
• Streptomyces somaliensis
• associated with actinomycotic
mycetoma
• Streptomyces anulatus (formerly
Streptomyces griseus)
• specimens has been increasingly isolated
from many clinical specimens, including
sputum, wound, blood, and brain.
 Other Actinomycetes
• Gordonia
• Aerobic
• Rhodococcus
• catalase positive • Rhodococcus equi
• gram positive to gram variable • found in soil and causes
• partially acid fast respiratory tract
• nonmotile. infections in animals.
• grow with mycelial forms that fragment • Human infection is rare
• demonstrate filaments,
into rod-shaped or coccoid elements—
hence the term ____________________
• They differ from rapidly growing some with branching.
mycobacteria by their weak acid
fastness and the absence of
• partially acid fast or acid
arylsulfatase fast.
 Other Actinomycetes
• Tropheryma whipplei
• agent of _____________________
• facultative intracellular pathogen
first identified in 1991 by using
PCR from a duodenal biopsy
specimen.
• gram-positive actinomycete
• detected in human feces, saliva,
and gastric secretions and is
apparently ubiquitous in the
environment.
• Rhodococcus
• Rhodococcus equi
• found in soil and causes
respiratory tract
infections in animals.
• Human infection is rare
• demonstrate filaments,
some with branching.
• partially acid fast or acid
fast.
 Spore-Forming, Nonbranching, Catalase-
Positive Bacilli

• Bacillus
 Bacillus

GENERAL CHARACTERISTICS • Bacillus anthracis

• gram positive or gram variable • Virulence Factors
• aerobic or facultative anaerobic bacilli • Capsule
that form endospores • Anthrax toxin

• some are thermophiles that grow best • consists of three proteins:

at 55° C or higher • _____________________________

• _____________________________
• _____________________________
 Bacillus
• Bacillus anthracis
• CLINICAL INFECTIONS
A. ANTHRAX
• CUTANEOUS ANTHRAX
• occur when wounds are
contaminated with anthrax spores
acquired through skin cuts,
abrasions, or insect bites.
• ____________________________
____________________________
____________________________
• INHALATION ANTHRAX
• _____________________________________
• acquired when spores are inhaled into the
pulmonary parenchyma.
• GASTROINTESTINAL ANTHRAX
• occurs when the spores are inoculated into a
lesion on the intestinal mucosa after
ingestion of the spores.
• INJECTIONAL ANTHRAX
• characterized by soft tissue infection
associated with “___________________” or
other forms of injection drug use and results
from the direct injection of the spores into
tissue.
 Bacillus anthracis
• LABORATORY DIAGNOSIS
• LABORATORY DIAGNOSIS • Cultural Characteristics
• Large,square-ended, gram-positive or • SBA: nonhemolytic, large, gray,
gram-variable rod found singly or in chains and flat with an irregular
• appearance of ______________________. margin because of outgrowths
• As the bacteria are subcultured, capsule of long, filamentous projections
production ceases.
• “________________________”
• Incubation in an atmosphere containing
increased CO2 can stimulate capsule • “________________________”
production
 Bacillus anthracis
• LABORATORY DIAGNOSIS
• IDENTIFICATION
• catalase positive
• nonmotile
• grows aerobically or anaerobically
• ferments glucose
• produces lecithinase
• opaque zone can be seen around
colonies growing on egg-yolk agar.
• high-salt (7% sodium chloride) and
low pH
• susceptible to penicillin (10 U/mL)
• Capsule production can be
detected by India ink staining on
blood or CSF
 Bacillus cereus
• GENERAL CHARACTERISTICS
• common cause of food poisoning
and opportunistic infections in
susceptible hosts
• grown aerobically at 37° C on SBA
• β-hemolytic frosted glass–appearing
colony
• spore-forming
• gram-positive bacilli
• Motile
• able to ferment salicin, and
lecithinase positive
Other Bacillus Species
• Bacillus subtilis
• Bacillus licheniformis
• Bacillus circulans
• Bacillus pumilus
• Bacillus sphaericus