Transcript Bordetella, Francisella, and Brucella “Those Gram-negative bacilli that have no family designation” Bordetella  Classification – the genus contains three medially important species     B.

Bordetella, Francisella, and
Brucella
“Those Gram-negative bacilli that
have no family designation”
Bordetella
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Classification – the genus contains three
medially important species
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B. pertussis
B. parapertussis
B. bronchoseptica
Morphology and cultural characteristics
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Small g-cb
B. parapertussis and B. bronchoseptica both grow
on sheep BA (SBA) in 1-2 days
Bordetella
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B. pertussis for initial isolation (The best clinical
specimen is a nasopharyngeal swab.) the organism
requires special media with additional nutrients for
growth and absorbents to remove toxic substances
found in complex media such as fatty acids and
sulfides.
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Borget-Gengou media – contains glycerol, potato infusion,
albumin (binds fatty acids), and up to 50% defibrinated
SRBCs
Charcoal agar supplemented with 10% horse blood with or
without cephalexin.
May take 3-7 days for growth and colonies are smooth,
raised, and glistening (phase 1 colonies).
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They are also hemolytic and produce toxin.
Charcoal-horse blood agar
Bordetella
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Upon extensive subculturing, the colonies become rough
(they progress through phases 2, 3, and finally 4) and can
now be grown on SBA.
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They are now less virulent due to loss of capsule, hemolytic
activity, and toxin production.
These changes, however, are reversible.
The organisms are strict aerobes and grow best at
35-370 C.
Biochemistry
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Nonfermentative
Use glucose and lactose oxidatively
B. bronchoseptica is motile, others are nonmotile
B. pertussis is – for urease, others are +
Bordetella
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No growth on Mac for B. pertussis, others are
variable
Oxidase test is variable
Virulence factors (B. pertussis)
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Pili for attachment
Pertactin, an outer membrane protein also acts as
an adhesion
Filamentous hemagglutinin – is found on the cell
surface of and is also secreted.
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It attaches to cilia by binding to exposed lactose
receptors.
Bordetella
Bordetella
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Pertussis toxin
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Secreted by type IV secretion system
Has one A subunit (toxic part), plus four different kinds of B
subunits (involved in binding).
Structure of pertussis toxin
A subunit
B subunits
Activation of pertussis toxin
Bordetella
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Once intracellular, the A subunit ADP ribosylates a critical
cysteine residue on the Gi regulatory proteins involved in
control of host cell adenylate cyclase resulting in
increased intracellular cAMP.
This causes cellular dysfunction.
Bacterial adenylate cyclase – is secreted and
inserts into the host cell membrane and is activated
by intracellular host cell calmodulin causing a
further increase in the intracellular levels of cAMP.
Increases in cAMP
Bordetella
The increase in cAMP from the combined
effects of pertussis toxin and bacterial
adenylate cyclase is associate with an
inhibition of host cell phagocytic cell
oxidative responses and the inhibition of
natural killer cell activity.
 Dermonecrotic toxin – is bacterial cell
associated and is released upon cell lysis
causing strong vasoconstrictive effects.
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Bordetella
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Trachael cytotoxin – is related to the
B.pertussis peptidoglycan.
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When this is incubated with cells in culture, the
cells are destroyed, so it might contribute to the
killing and sloughing off of ciliated cells in the
respiratory tract.
Lipooligosaccharide associated with the
surface of the bacteria and has potent
endotoxin activity.
Bordetella
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Clinical significance
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B. pertussis – causes whooping cough
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Acquired by inhalation of droplets containing the organism
The organism attaches to the ciliated cells of the
respiratory tract.
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During an incubation period of 1-2 weeks, the organism
multiplies and starts to liberate its toxins.
Next the catarrhal stage occurs - the patient has a mild
cough and sneezing whereby large numbers of organisms
are spread through the respiratory secretions.
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This last ~ 2 weeks.
Bordetella
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Next is the paroxysmal stage that lasts 4-6 weeks.
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The patient has rapid, consecutive coughs with a rapid
intake of air between the coughs (has a whooping sound).
The ciliary action of the respiratory tract has been
compromised, mucous has accumulated, and the patient is
trying to cough up the mucous accumulations.
The coughs are strong enough to break ribs!
Other symptoms due to the activity of the released toxins
include:
Increased peripheral lymphocytes due to a blocking of
homing of lymphocytes to the spleen and lymph nodes.
Metabolic alteration such as increased insulin release
and the resulting hypoglycemia
Increased capillary permeability and increased
susceptibility to histamine, serotonin, and endotoxin
shock
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Bordetella
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Finally there is a convalescent stage during
which symptoms gradually subside.
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This can last for months.
B. pertussis rarely spreads to other sites, but a
lot of damage may occur, such as CNS
dysfunction which occurs in ~10 % of the cases
and is due to an unknown cause.
 Secondary infections such as pneumonia and
otitis media are common.
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B. pertussis pathogenesis
Bordetella
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B. parapertussis – causes a mild form of whooping
cough
B. bronchoseptica
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Widespread in animals where it causes kennel cough.
Occasionally causes respiratory or wound infections in
humans.
Treatment
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Erythromyin – only effective in early stages of the
disease before the toxin(s) have been released
Vaccination P part of DPT (killed, encapsulated
organism); a subunit vaccine has also been
developed (purified pertussis toxin).
Francisella
Classification – only 1 pathogenic species – F.
tularensis
 Morphology and cultural characteristics
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Minute, pleomorphic g- rod that stains poorly
Staining may be bipolar
Nonmotile
Nonencapsulated
Won’t grow on ordinary media – requires cysteine
or cystine for growth
Francisella
Grow on blood-glucose-cysteine agar
 Will also grow on Chocolate or MTM with
added isovitalex
 Colonies may grow in 24 hours or may take
5-7 days for growth
 Is a strict aerobe
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Biochemistry
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Oxidase -
Francisella
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No glucose fermentation
Won’t grow on Mac
Diagnosis
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Is best done by showing an antibody titer increase
of 1:40 in a patient not previously infected.
Culturing the organism is hazardous and should
only be done under a biosafety hood.
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The organism is highly contagious and the infective dose
for an aerosol route of infection is very small.
Francisella
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Clinical significance – tularemia is a disease
mostly in rabbits and other rodents.
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It is usually transmitted to man through skin
abrasions after exposure to infected animals or by
ticks or deer flies that have fed on infected rodents.
It can also be acquired by inhalation or ingestion.
The manifestations of disease depend upon the
mode of entry:
Francisella
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Entry through skin abrasions (ulceroglandular form
of the disease) - after ~ 48 hours a lesion occurs at
the inoculated site.
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It forms an ulcer and the patient may have headaches,
pain and fever as adjacent lymph nodes become enlarged.
If not contained, this can progress to septicemia,
pneumonia, and abscesses throughout the body.
The organism survives for long periods of time inside
phagocytic cells).
Skin lesion
Francisella
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Ingestion (typhoidal form of the disease) – the
focus of infection is the mouth, throat, and GI tract.
Inhalation (pneumonic form of the disease) – This is
the most severe form of the disease and it
manifests as a pneumonia with a high mortality rate
of 30% in untreated cases.
Antimicrobial susceptibility
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Streptomycin or tetracycline
An attenuated, live vaccine that protects against the
inhalation form of the disease is available for those
exposed to the organism.
Brucella
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Classification
Are all intracellular organisms
 4 species can infect humans
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B. abortus
 B. suis
 B. melitensis
 B. canis
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Morphology and cultural characteristics
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Small g-cb that stain poorly
Brucella
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Nonmotile
Nonencapsulated
In tissues are found intracellularly
Most clinical isolates come from blood cultures
(Castenada’s media which has both a solid and a
liquid phase)
Requires enriched media containing meat infusion
or tryptone
Will grow on CBA or chocolate agar
Growth is slow and may take 72 hours
Colonies start as tiny pinpoint, translucent colonies
that become gray with age.
Brucella
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B. abortus requires 10% CO2 for growth, others do
not
Biochemistry
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Oxidase +
Nonfermentative
Urease +\catalase +
H2S produced by B. abortus and B. suis
Speciated based on the ability to grow in the
presence of the dyes basic fuchsin and thionine
Brucella
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Antigenic structure
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2 antigens that are part of the LPS are recognized:
A and M
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Virulence factors
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B. melitensis has the highest concentration of M and
causes the most serious infections
Endotoxin
Clinical significance
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Has a tropism for erythritol
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Animal fetal tissues and placenta, other than those in
humans, are rich in erythritol and, therefore, the organisms
often cause abortions in these animals.
Brucella
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Causes Brucellosis or undulent fever in man
following ingestion of contaminated milk or cheese
from goats (B. melitensis), cows (B. abortus), pigs
(B. suis), or canines (B. canis).
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Man can also acquire the organism via contact with
infected animals.
Clinical manifestations range from subclinical, to chronic
with low grade symptoms of low fever and muscular
stiffness, to acute with fever and chills.
The fever typically spikes each evening and this coincides
with the release of organisms from phagocytes (hence the
name undulent fever).
The patient may also experience malaise, weakness,
enlarged lymph nodes, weight loss, and arthritis.
Brucella
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Antibiotic susceptibility
Chemotherapy is difficult because of the
intracellular survival of the organism.
 Tetracycline for 21 days, sometimes
combined with streptomycin.
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