Gram positive bacilli
Bacillus spp.
Corynebacterium diphteria
Erysiphelothrix
Listeria spp.
Gram positive aerobic bacilli (2 hours):
Learning Objectives
• 1.
Defines ‘’Gram positive aerobic bacilli’’
• 1.1 Lists Gram positive aerobic bacilli in normal flora.
• 1.2. Lists pathogenic Gram positive aerobic bacilli for human.
• 1.3. Lists virulance factors, defines tissue damage mechanisms.
2.
Lists the clinical tables related with Gram positive aerobic bacilli
and defines pathogenetic mechanisms.
•
• 2.1. Defines the clinical importance of Gram positive aerobic bacilli
(Bacillus spp., Corynebacterium diphteria,Erysiphelothrix spp, Listeria
spp.)
• 2.2. Lists the classical culture methods, microscopy and additional
diagnostic methods (Schick test, ELEK test)
• 2.3. Lists the bioterrorism Gram positive aerobic bacilli agents.
Bacillus spp
• After coagulase-negative staphylococci and
diphtheroids, members of the genus Bacillus are the
third most common skin contaminant found in clinical
specimens.
• On Gram stain they are large, wide, Gram-positive
rods, often occurring singly or in pairs, which can
produce endospores.
• They may be confused with clostridia on direct stains
from specimens.
Bacillus spp-II
• Usually they are readily distinguished from lactobacilli
which tend to occur as long, narrow Gram-positive
rods which often chain. Also, Bacillus spp. are
catalase-positive, while lactobacilli are not
• Colony morphology of Bacillus spp. is highly variable,
often growing as large to very large gray-white
colonies that may be dry in appearance, or which
may produce a contiguous mat of wet, blistery
colonies.
Bacillus spp-III
• a genus of Gram-positive bacilli which are commonly
found in nature
– soil,
– water,
– airborne dust.
• members of natural flora in the human intestines.
• Most species of Bacillus are harmless saprophytes,
• two species are considered medically significant:
– B.anthracis
– B. cereus.
B. anthracis
• causes anthrax in cows, sheep, and sometimes humans.
• Anthrax is transmitted to humans via
– direct contact with animal products
– inhalation of endospores.
– ingestion
• Sources of infection are usually industrial or agricultural and the
infection is classified as one of three types:
 
 
 
CUTANEOUS INFECTION (95% of human cases)
INHALATION ANTHRAX (rare) : Bioterrorism agent
GASTROINTESTINAL ANTHRAX (very rare!)
Diagnosis
Microscopy:
• Gram positive centrally located spore forming bacilli
• In tissue imprints
– Gram positive capsulated chain forming bacilli
Culture:
Blood agar
Bacillus produces large, spreading, gray-white colonies with
irregular margins: Medusa head
A unique characteristic of this bacterium is its ability to produce
endospores when environmental conditions are stressful.
The only other known spore-producing bacterium is Clostridium
Diagnosis-II
•
Typical skin lesion: Dark centered necrotic lesion
•
Microscopy from the skin lesion: (Specimen should be taken at the margin
of healthy and diseased tissue)
– Under the microscope, B. anthracis cells appear to have square ends
and seem to be attached by a joint to other cells.
– The spores are best observed when the bacterium is cultured on
artificial media.
•
•
Culture
Blood agar: Medusa head-like colonies
•

LABORATORY INDICATIONS:
Nonhemolytic (sheep blood agar)

Non-motile


Gel hydrolysis Catalase +
B. cereus
• Unlike B. anthracis, B.cereus is a motile bacterium
• cause toxin-mediated food poisoning.
• It is known to inhabit many kinds of food
– Rice
– stew,
– cereal,
– milk .
• The two toxins released by the vegetative form of the bacilli
– vomiting
– diarrhea, (symptoms similar to those of Staphylococcus food
poisoning).
• Because toxin production usually takes place after the infected
foods are cooked, proper cold storage of food is recommended
immediately after preparation.
Bacillus subtilis grown in air on sheep blood trypticase soy agar
Bacillus cereus
Bacillus anthracis
Bacillus anthracis
Evolution of an anthrax eschar in a 4-year-old boy. (A&B) the
lesion when first seen (day 0). Note the arm swollen from the
characteristic edema.
(C) Day 6
(D) Day 10. (E) Day 15. Although penicillin treatment was
begun immediately and the lesion was sterile by about 24
hours, it continued to evolve and resolve as seen
The pathogenicity of B anthracis depends on two virulence factors:
•a poly-y-D-glutamic acid polypeptide capsule, which protects it from
phagocytosis by the defensive phagocytes of the host,
•a toxin produced in the log phase of growth.
This toxin consists of three proteins:
•protective antigen (PA) (82. 7 kDa),
•lethal factor (LF) (90.2 kDa),
•edema factor (EF) (88.9 kDa).
Colonies of B anthracis on a blood agar plate. Note the characteristic
tackiness of colonies that allow them to be teased upright with a loop
(foreground) and the characteristic tailing seen in the background
(arrows).
Corynebacterium diphtheriae
• aerobic
• extracellular
• rods; club-shaped, has granules “V”, “L”, or “Chinese
Letter”
colonies = Dark gray or black on potassium tellurite
medium
C. diphtheriae-II
• no motility
• no capsule & glycocalyx
• exotoxins = diphtheria toxin
– An ADP-Ribosyltransferase
• Peptide B binds to host cells to transport
peptide A inside
• Peptide A has the enzymatic activity
• attaches ADP ribose and prevents
ribosome movement along mRNA
• blocks host EF2 (a protein synthesis
elongation factor tRNA translocase)
• blocks protein synthesis
• can kill host’s NK cells (natural killer)
• coded on viral DNA which gets
integrated into the bacteria by lysogeny
– symptoms:
pseudomembrane formation in the throat;
exudate forms a tough gray membrane
which can lead to stridor (high pitched
respiratory sound), respiratory distress,
cyanosis, lymphadenopathy
– can be fatal
pseudomembrane
• intoxication consequences of diphtheria
toxin
– cardiac toxicity - occurs weeks after initial
infection
– myocarditis, arrhythmias - Can be fatal
– neurologic toxicity - occurs only following
severe infection - Early (first few days) –
paralysis of soft palate and pharynx
• later (months later) - peripheral motor
neuropathy
• diagnosis must be made fast, and is
based solely on symptoms
• skin infections are rare
– Infects an open wound, mostly in persons
with poor hygiene - tropics - presents
with gray membrane on non-healing wound
• Schick Skin Test (rarely performed)
– Positive sign is if there is no reaction to
toxin when injected intradermally - means
that patient is immune
• Humans are the only reservoir for C.
diphteria. Upper respiratory tract
infections and skin lesions
• Horizontal transmission occurs via
respiratory droplets
virulance factors
• Diphtheria exotoxin
• Storage granules (metachoromatic
bodies)
– Contain phosphate polymers for highenergy reserve
– Stain with metachromatic dye : methylene
blue
• Growth on Loeffler medium
• Metachromatic staining of storage
granules
• Growth on tellurite medium - black
colonies
In vitro toxigenicity test
Elek Test
• In a strip of sterile filter paper antitoxin is
impregnated
• A heavy inoculum is streaked on a agar surface
• Antitoxinated paper is placed on the surface of the
agar medium at right angles to the inocula.
• Allowed to incubate for 24 hours.
• If the organisim is toxigenic a visible line of Ag-Ab
precipitate will form
Treatment
• Penicillin G to kill the organism
• Erythromycin in penicillin allergy
• Diphtheria antitoxin (horse or humanderived antibodies to the toxin) must be
administered immediately
VACCINE & TOXOID
• Toxoid - inactive toxin is given as part
of the DPT vaccine
ERYSIPELOTHRIX
• E. rhusiopathiae, the only species of this genus,
• been found in many farm animals such as pigs, horses, and
turkeys
• is better known as a veterinary pathogen than as a human
pathogen. When cultured on blood agar or some other nutrient
medium, Erysipelothrix forms notably large colonies.
• Occasionaly, it can infect a human host and cause an
inflammatory skin disease, Erysipeloid.
• Treatment usually consists of penicillin G, ampicillin, or
cephalothin.
• LABORATORY INDICATIONS:
 Catalase -
 Non-motile
 TSI : H2S +
LISTERIA
• Gram-positive rod which is not capable
of forming endospores.
• several species exist, but two species
are important for human pathogenic
significance:
• L. monocytogenes
• L. ivanovii.
Listeria monocytogenes
• normal inhabitant of the gastrointestinal tract and of animal
feces
• Found responsible in several food poisoning epidemics.
– led to a 1986 outbreak in Massachusetts hospital patients.
– Those infected suffered from vomiting, nausea, and diarrhea.
– the hospital patients contracted the microbe from the infected
hospital food and were at high risk of infection.
• High risk group:
– newborns,
– pregnant women and their fetuses,
– the elderly,
– immune compromized
Clinical findings:
•
•
•
septicemia
meningitis
listeriosis: is an inflammation of the brain.
LABORATORY INDICATIONS:

Catalase +

Motile at room temperature
• Growth at 4 degrees Celsius
• Bile esculin hydrolysis
• Beta-hemolysis
Treatment:
•
Antibiotics are recommended for treatment of infection
•
most strains of Listeria are sensitive to ampicillin and gentamicin