Bordetella, Francisella, and Brucella

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Bordetella, Francisella, and Brucella

• Those Gram-negative bacilli that have no family
  designation

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Bordetella

• Classification the genus contains three
  medially important species
• B. pertussis
• B. parapertussis
• B. bronchoseptica
• Morphology and cultural characteristics
• Small g-cb
• B. parapertussis and B. bronchoseptica both grow
  on sheep BA (SBA) in 1-2 days

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Bordetella

• 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.
• 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).
• They are also hemolytic and produce toxin.

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Charcoal-horse blood agar
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Bordetella

• Upon extensive subculturing, the colonies become
  rough (they progress through phases 2, 3, and
  finally 4) and can now be grown on SBA.
• 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
• Nonfermentative
• Use glucose and lactose oxidatively
• B. bronchoseptica is motile, others are nonmotile
• B. pertussis is for urease, others are

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Bordetella

• No growth on Mac for B. pertussis, others are
  variable
• Oxidase test is variable
• Virulence factors (B. pertussis)
• 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.
• It attaches to cilia by binding to exposed
  lactose receptors.

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Bordetella
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Bordetella

• Pertussis toxin
• Secreted by type IV secretion system
• Has one A subunit (toxic part), plus four
  different kinds of B subunits (involved in
  binding).

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Structure of pertussis toxin
A subunit
B subunits
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Activation of pertussis toxin
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Bordetella

• 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.

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Increases in cAMP
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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

• Trachael cytotoxin is related to the
  B.pertussis peptidoglycan.
• 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.

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Bordetella

• Clinical significance
• B. pertussis causes whooping cough
• Acquired by inhalation of droplets containing the
  organism
• The organism attaches to the ciliated cells of
  the respiratory tract.
• 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.
• This last 2 weeks.

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Bordetella

• Next is the paroxysmal stage that lasts 4-6
  weeks.
• 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

• Finally there is a convalescent stage during
  which symptoms gradually subside.
• 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
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Bordetella

• B. parapertussis causes a mild form of whooping
  cough
• B. bronchoseptica
• Widespread in animals where it causes kennel
  cough.
• Occasionally causes respiratory or wound
  infections in humans.
• Treatment
• 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).

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Francisella

• Classification only 1 pathogenic species F.
  tularensis
• Morphology and cultural characteristics
• Minute, pleomorphic g- rod that stains poorly
• Staining may be bipolar
• Nonmotile
• Nonencapsulated
• Wont grow on ordinary media requires cysteine
  or cystine for growth

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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
• Biochemistry
• Oxidase -

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Francisella

• No glucose fermentation
• Wont grow on Mac
• Diagnosis
• Is best done by showing an antibody titer
  increase of 140 in a patient not previously
  infected.
• Culturing the organism is hazardous and should
  only be done under a biosafety hood.
• The organism is highly contagious and the
  infective dose for an aerosol route of infection
  is very small.

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Francisella

• Clinical significance tularemia is a disease
  mostly in rabbits and other rodents.
• 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

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Francisella

• Entry through skin abrasions (ulceroglandular
  form of the disease) - after 48 hours a lesion
  occurs at the inoculated site.
• 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).

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Skin lesion
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Francisella

• 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
• Streptomycin or tetracycline
• An attenuated, live vaccine that protects against
  the inhalation form of the disease is available
  for those exposed to the organism.

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Brucella

• Classification
• Are all intracellular organisms
• 4 species can infect humans
• B. abortus
• B. suis
• B. melitensis
• B. canis
• Morphology and cultural characteristics
• Small g-cb that stain poorly

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Brucella

• Nonmotile
• Nonencapsulated
• In tissues are found intracellularly
• Most clinical isolates come from blood cultures
  (Castenadas 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.

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Brucella

• B. abortus requires 10 CO2 for growth, others do
  not
• Biochemistry
• 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

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Brucella

• Antigenic structure
• 2 antigens that are part of the LPS are
  recognized A and M
• B. melitensis has the highest concentration of M
  and causes the most serious infections
• Virulence factors
• Endotoxin
• Clinical significance
• Has a tropism for erythritol
• Animal fetal tissues and placenta, other than
  those in humans, are rich in erythritol and,
  therefore, the organisms often cause abortions in
  these animals.

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Brucella

• 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).
• 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.

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Brucella

• Antibiotic susceptibility
• Chemotherapy is difficult because of the
  intracellular survival of the organism.
• Tetracycline for 21 days, sometimes combined with
  streptomycin.