Module 10 Lecture

  • classification

    • Domain: bacteria

    • Phylum: proteobacteria

    • Class: B-proteobacteria

    • Order: Burkholderiales

    • Family: Alcaligenaceae

    • Genus: Bordetella

    • Species: B. pertussis, B. parapertussis, B. bronchiseptica

    • genome — 4.1 megabases, ~3800 genes, 68% GC content

  • B. pertussis general characteristics

    • small gram-negative coccobacilli, in singles or pair arrangement

    • 0.2 × 0.7 um

    • non-motile

    • non-spore forming

    • on charocal-horse blood agar, colony morphology — “mercury drops”

      • pathogen is senistive to fatty acids, metal ions, and sulfides

      • needs supplemental protectant such as charcoal in the media

      • smooth, convex, small, silvery/gray color

    • slow growing; may take 3-6 days for colonies to form

    • B. parapertussis has slightly larger colonies, dull, faster growing

    • aerobe

    • catalase positive

    • oxidase positive

    • fastidious; very picky — does not growth on standard growth media

    • unique LPS; different phosphate content (variable phosphate content)

    • reservoir in respiratory ciliated epithelium

      • found in humans & primates

  • B. pertussis disease and virulence factors

    • disease of upper respiratory tract known as whooping cough or pertussis

      • intense coughing fits followed by gasp of air

      • transmission via respiratory droplets through fomites or direct contact; very communicable

      • in adults - milder

      • in children - severe (usually due to vaccination status)

      • B. parapertussis does cause a very mild form of pertussis & B. bronchiseptica causes kennel cough

    • virulence factors of pertussis

      • filamentous hemagglutinin toxin (FHA); long filamentous structure on outside of the bacteria which promotes adhesion of ciliated epithelia cells of the upper respiratory tract

        • ciliary stasis — can bind two cilia and prevent the sweeping motion of cilia

      • pertussis toxin (PT); extracellular

        • found at the end of FHA & outside of the bacteria

        • A-B toxin

          • one A subunit and 5 B subunits (B subunits facilitates attachment to specific cell types & A subunit has an enzymatic function

          • B subunit effects ciliated cells & phagocytic cells; when bound it transfers A subunit into host cell

          • A subunit ADP ribsosylate

            • G protein inhibition (increase in cAMP)

            • reverse flow of ions — Cl ions and water moving out to result in accumulation of mucus and fluid on the outside of the cells

            • in phagocytotic cells disruption of chemotaxis (phagocytotic behavior of moving towards pathogen and engulfing)

      • invasive adenylate cyclase

        • AB toxin

        • B subunit targets phagocytic cells

        • A subunit is an adenylate cyclase (increase cAMP)

          • disrupts normal phagocytic activity

      • tracheal toxin

        • not a protein; peptidoglycan fragment

        • kills ciliated epithelia cells & cause extrusion from the epithelia layer (damages tissue)

      • dermonecrotic toxin

        • causes inflammation & local necrosis (damages tissue)

    • damages tissue in respiratory tract and the ciliated epithelia cells that evict pathogens & fluid extruded from adenylate cyclase and pertussis toxin

  • Whooping cough (pertussis)

    • after exposure, symptoms after 7-10 days

    • catarrhal phase in first week

      • bacteria present, easy to culture from infected individual, good response to antibiotic treatment, very contagious at this stage

      • mild symptoms at this phase such as cold and runny nose

    • paroxysmal phase in six weeks

      • bacteria are harder to culture from infected individuals, not good response to antibiotics, violent coughing fit which triggers vomiting, high incidence of secondary infections

        • not enough oxygen = brain damages / seizures

    • convalescent phase (recovery phase)

      • fewer coughing fits in frequency

  • Lab diagnosis of B. pertussis

    • taking patient history of intense prolonged coughing fits and vaccination status (DTaP vaccination)

    • specimen of nasopharyngeal swap dipped in penicillin to reduce indigenous flora at that body site

      • culture & grow on a media with a protectant

      • plates used: Bordet-Gengou & charcoal-horse blood agar

        • BG media has starch for protectant component; shiny, tan, smooth, convex, tiny colonies

        • charcoal plate has charcoal for protectant and colonies appear as “mercury drops”

      • gram stain as gram-negative tiny coccobacilli

    • to distinguish between B. parapertussis on media — dull, a bit larger, and faster growth (2-4 days for growth)

    • PCR

      • no single target gene; identifiers as presence of B. pertussis is pxtS1 gene (pertussis toxin) & IS431

        • in later stages of disease, the bacteria are not as abundant so culturing is less likely to occur; more likely to ID by PCR in later stages

    • direct fluorescent antibody detection

      • antibodies generated against antigen of the bacteria

  • treatment and prevention for B. pertussis

    • antibiotic prescription —most effective in catarrhal phase (early infection)

      • shortens course of infection & reduce overall symptoms

      • prophylactic treatment for those in close contact with those diagnosed with pertussis, regardless of vaccination status (infection control method)

    • cough suppressant

    • respiratory support for young children / infants

    • vaccination — combination DTaP (diphtheria, tetanus, acellular pertussis + FHA & PT)

      • effectiveness of vaccine is decent; immunity is not lasting as long as it had in the past