Microbiology Quiz: Neisseria and Meningitis

Neisseria, Haemophilus and Bordetella

• These are gram-negative bacteria.

• Neisseria:

  • Anaerobic
  • Gram-negative cocci, 0.6-1 µm in size
  • Arranged in pairs with flattened sides
  • Non-motile
  • Catalase positive
  • Oxidase positive
  • Oxidize carbohydrates (do not ferment)
  • Sugar used in presence of O2 to produce acid
  • Some are normal flora of the upper respiratory tract
  • Others are obligate human pathogens
  • Two medically relevant species: N. meningitidis and N. gonorrhoeae
    • N. meningitidis causes meningitis (nasopharynx colonization, but not always disease)
    • N. gonorrhoeae causes gonorrhea (a common STD)
  • N. gonorrhoeae is fastidious, requiring cysteine and lack of fatty acids in the growth medium, has an optimum growth temperature, and is difficult to grow. Easy to transmit person-to-person.
  • Typical gram-negative: thin peptidoglycan + outer membrane
  • LPS of Neisseria is called lipooligosaccharide (LOS)
  • High branched sugar chains (oligosaccharides). Does lipid A have endotoxic effect?
  • N. meningitidis: covered in carbohydrate capsule; N. gonorrhoeae: no true capsule, but sugars with negative charge on surface
  • Surface and membrane proteins:
    • Pili: attachment and transfer of genetic material.
    • Porin: forms pores and channels in the outer membrane
    • Rmp protein: antibodies interfere with other antibodies, yet antibodies may help pathogenesis
  • Requires iron.
  • Produce IgA protease: inactivates IgA.
  • Surface proteins mediate attachment and penetration of host cells
  • Meningococcal pili preferentially bind cells in nasopharynx
  • N. gonorrhoeae: internalized by phagocytes and survives
  • LOS from both pathogens stimulates inflammation; IgG is the primary antibody produced against N. gonorrhoeae, also targeting Omp's, LOS, pili, and capsule
  • Protection against meningococci: humoral or cellular?
  • Meningitis occurs when antibody response is low (ex. children), and some infants are initially protected.
  • N. meningitidis (a.k.a. the “meningococcus”): can be carried without symptoms, has a large antiphagocytic capsule, and is very sensitive to temps above or below 37˚C. It can undergo autolysis. Colonizes the nasopharynx. Meningococcal meningitis (inflammation of meninges) and meningococcemia (characterized by skin lesions) cause of these illnesses in humans. Can survive in immune cells.
  • Meningitis: mildest form, fever and malaise, resolving in 1-2 days; fulminant form is fever, vomiting, headache, seizure, coma. Infants have initial symptoms of fulminant meningitis. Progressive neurological damage, convulsions, spinal rigidity, hamstring spasms, can kill. Meningococcemia: septicemia but no meningitis.
    • Haemorrhages of skin (30% to 60% of patients), pulmonary insufficiency, death in 24 hours possible.

• Haemophilus:

  • Gram-negative rods: outer membrane with LPS (endotoxin), strain-specific proteins.
  • Non-spore formers
  • Non-motile
  • Aerobic or Facultative anaerobes
  • Fastidious
  • Requires hemin (X factor) and NAD (V factor). Must be heated first.
  • Some have a capsule (6 different serotypes). Non-encapsulated still cause disease
  • 1 serotype vaccine: type B
  • Commonly colonize mucous membranes of upper respiratory tract (healthy): H. parainfluenzae and nonencapsulated H. influenzae
    • H. parainfluenzae 10% of saliva flora
    • Local spread possible (otitis media, sinusitis, bronchitis, pneumonia)
  • Disseminated disease is rare
  • Most common disease causing serotype: H. influenzae serotype B
  • Uncommon in upper respiratory tract.
  • Vaccine available (1987) but limited to those 2 months of age and older
  • 20,000/year infections occurring in the developing nations 3 million case/year infection
    • Epidemiology: primarily affects unvaccinated children, similar symptoms of other types of bacterial meningitis, and 1-3 day initial mild symptoms of meningitis.
    • Recovery: with proper intervention, 90% have favorable outcomes
    • Epiglottitis, a life-threatening inflammation of epiglottis (cartilage structure).
    • Arthritis: prevalent before vaccine at <2 yrs of age and characterized by pain swelling in joints with inflammation.
    • Non-encapsulated: otitis, sinusitis and lower respiratory tract infections
    • Acute conjunctivitis, H. aegypticus: contagious inflammation of membrane lining eyelid and whites of eyes.
    • Chancroid (STD), H. ducreyi: genital sore that ulcerates (2 days)
  • Laboratory diagnosis: sample fluid (CSF), microscope (sensitive and specific), culture use supplements (chocolate or Levinthal's agar, Staphylococci); agglutination
  • Treatment/prevention: antibiotics (cephalosporins) vaccine (against H. influenzae type B - conjugate of pure capsular PRP); 3 doses before 6 months of age.

• Bordetella:

  • Gram-negative coccobacillus; strict aerobes
  • Simple nutritional requirements. Charcoal, starch, blood included to absorb any toxic compounds
  • Most important species: B. pertussis, major disease: whooping cough, exclusive human pathogen; vaccine available (DTP), Still ~60 million cases/year worldwide, 12,000 cases/US.
  • Distinguished from other Bordetella species by oxidase + test, urease - test, non-motile, and no growth on sheep blood agar.
  • B. pertussis inhaled into respiratory tract,
  • Binds to ciliated cells lining airways
  • Damage cell (3 toxins)
    1. Pertussis toxin, a two-component toxin, that enters cells to increase respiratory secretions/mucus.
    2. Tracheal toxin, directly interferes with DNA of ciliated cells, to inhibit cilia and eventually destroy ciliated cells.
    3. Hemagglutinin, promoting binding to membranes of ciliated cells by increasing mucus.
  • Disease progression: 7-10 day incubation after inhalation, 3 stages
    • Catarrhal: peak bacteria, disease may not yet be diagnosed (high infectivity)
    • Paroxysmal: coughing attacks, mucus clearance impaired, vomiting and exhaustion, and 40-50 paroxysms/day
    • Convalescent: cough stops, paroxysms subside, secondary complications possible
  • Specimen collection and diagnosis (to avoid drying/exposure to drying and cold temperatures): nasopharyngeal (no cotton swabs due to toxic fatty acids present), plate immediately in specialized transport media, use chocolate or Regan-Lowe charcoal agar, microscopy (fluorescent antibodies)
  • Treatment: Supportive therapy (antibiotics, macrolides (e.g., erythromycin, azithromycin), dosage inactivated pertussis toxin + bacterial component of vaccine); 1-5 doses; 2, 4, 6, 15 months and one between 4-6 yrs (DTaP), booster for adolescents and pregnant women
  • Recent outbreaks (2011-2012) due to elevated awareness (improved diagnostic testing, better reporting) and circulation of bacteria. Wiping immunity from vaccines. Infants and very young children - highest risk for serious illness. BUT: adolescent cases (13-14 yrs old) increasing.

• Legionella:

  • Gets name from the first outbreak of Legionnaires Disease in American Legion 1976.
  • Gram-negative, poorly staining
  • Pleomorphic coccobacilli rods (long/slender)
  • Obligate aerobes, nutritionally fastidious
  • Requires L-cysteine and iron and a buffered charcoal yeast extract agar.
  • Water, lakes, cooling towers / air conditioners, showers, hot tubs - often sources of contamination
  • Not spread human to human
  • L. pneumophila likely disease; causes Legionnaires disease and Pontiac fever (18,000 cases/year in US)
  • Affects free-living amoeba in nature and biofilms.
  • Infects alveolar macrophages, monocytes, and epithelial cells
  • Survives intracellularly by preventing lysosomal fusion (survives within body cells)
  • Legionnaires disease: 1-10 days, severe form of pneumonia, multiorgan infection
  • Pontiac fever: 1-2 days, high fever, chills, headache, no pneumonia
  • 15% death in healthy, 75% in immunocompromised.
  • Diagnostics:
    • Microscopy: poor staining, hard to find in tissue sections
    • Culture: buffered charcoal yeast extract agar; grow for 3-5 days
    • Media requires (L-Cysteine and iron)
    • Test growth with/without supplements
    • Sheep's blood agar, not helpful.
    • Serological testing (ex. Legionella urinary antigen test), for initial detection, useful
    • Nucleic acid amplification (ex. PCR) for respiratory samples and water
    • Antibody titers (useful to detect prior exposure)
  • Treatment: macrolides or fluoroquinolones
  • Prevention and screening: water cooling towers - tested by labs, water systems in hospitals/ nursing homes treated (hyperchlorination, heating, biocides, and coper-silver ionization)

• Corynebacterium:

  • Gram-positive rods; clumps, short chain (V or Y arrangement), irregular / club-shaped
  • Aerobic or facultative anaerobes, catalase +
  • Do not form spores
  • Unique cell wall (diaminopimelic acid and mycolic acid)
  • Stain with Methylene Blue (Metachromatic granules inside some bacteria)
  • Normal colonizers (skin, upper RT, GI, or urogenital)
  • Opportunistic pathogens
  • Pathogen: C. diphtheriae causes diphtheria.
  • Irregular-club shaped rods
  • Metachromatic granules visible after staining with methylene blue
  • Granules: inorganic polyphosphates (volutin); serve as energy reserves. Not membrane bound.
  • Two-component exotoxin, encoded by bacteriophage. Expressed when iron concentrations are low.
  • Enter target cells, stops protein synthesis in cell.
  • Phospholipase (D): increases permeability of blood vessels
  • Allows organism to spread within the nasopharynx
  • Epidemiology: asymptomatic carriage in throat, spread via respiratory droplets.
  • Sore throat, low-grade fever may occur. Grey pseudomembrane over throat.     - Bacteria, lymphocytes, plasma cells, fibrin, and dead cells
  • Toxin causes vessel damage, bleeding, myocarditis, trouble breathing; spreads through blood (body); death can occur from toxin-mediated heart failure
  • Diagnostics:
  • Microscopy: methylene blue stain (showing metachromatic granules); Gram stain for Gram-positive pleomorphic rods arranged in irregular formation.
  • Culture: cysteine or serum tellurite agar (gray to black colonies). Tellurite in media, which is reduced intracellularly to tellurium.
  • Differentiated from Arcanobacterium and C. pseudotuberculosis (box 27.1): testing for the presence of DAP (diaminopimelic acid) in the cell wall.