Bordetella, Francisella, and Brucella

“Those Gram-negative bacilli that have no family designation”

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

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.

Charcoal-horse blood agar

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 +

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.

Bordetella

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).

Structure of pertussis toxin

A subunit

B subunits

Activation of pertussis toxin

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.

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.

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.

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.

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

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.

B. pertussis pathogenesis

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).

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 Won’t grow on ordinary media – requires cysteine

or cystine for growth

Francisella

Grow on blood-glucose-cysteine agarWill also grow on Chocolate or MTM with

added isovitalexColonies may grow in 24 hours or may take

5-7 days for growth Is a strict aerobe

BiochemistryOxidase -

Francisella

No glucose fermentation Won’t grow on Mac

Diagnosis 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. The organism is highly contagious and the infective dose

for an aerosol route of infection is very small.

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:

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).

Skin lesion

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.

Brucella

Classification Are all intracellular organisms4 species can infect humans

B. abortusB. suisB. melitensisB. canis

Morphology and cultural characteristicsSmall g-cb that stain poorly

Brucella

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

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

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.

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.

Brucella

Antibiotic susceptibilityChemotherapy is difficult because of the

intracellular survival of the organism.Tetracycline for 21 days, sometimes

combined with streptomycin.