Bordetella, Francisella, and Brucella

1. Bordetella, Francisella, and Brucella “Those Gram-negative bacilli that have no family designation”

2. 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

3. 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.

4. Charcoal-horse blood agar

5. 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 +

6. 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.

7. Bordetella

8. 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).

9. Structure of pertussis toxin A subunit B subunits

10. Activation of pertussis toxin

11. 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.

12. Increases in cAMP

13. 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.

14. 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.

15. 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.

17. 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

18. 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.

19. B. pertussis pathogenesis

20. 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).

21. 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

22. 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 -

23. 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.

24. 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:

25. 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).

26. Skin lesion

27. 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.

28. 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

29. 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.

30. 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

31. 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.

32. 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.

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