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Corynebacterium listeria erysipelothrix_bacillus

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Corynebacterium listeria erysipelothrix_bacillus

  1. 1. Laboratory diagnosis of infections caused by Gram positive bacilli Corynebacterium, Listeria, Erysipelothrix, Bacillus
  2. 2. Gram positive and Gram negative bacilli Genera: • Corynebacterium • Listeria • Erysipelothrix • Bacillus Family Enterobacteriaceae Genera: A. Highly pathogenic: • Yersinia • Salmonella • Shigella A. Facultatively pathogenic: • E.coli • Klebsiella • Proteus • Enterobacter • Serratia • Citrobacter
  3. 3. Genus Corynebacterium Species Corynebacterium diphtheriae - high pathogenicity - Clinical significance: diphtheria = disease produced by the diphtheric toxine - → symptoms at the entry gate: sore throat, adherent membrane (pseudomembrane) on tonsils, pharynx, nasal cavity - → general toxic symptoms: fever (hematogenic difusion of diphtheric toxin)
  4. 4. Left: diphtheric pseudomembrane on tonsils Right: diphtheric skin lesion
  5. 5. Genus Corynebacterium Other pathogenic species (zoonosis) Producers of diphteric toxin: • Corynebacterium ulcerans – infection of cattle – mastitis; infection transmissible to humans via infected milk • Corynebacterium pseudotuberculosis – comensal of animals (horses, sheep) – transmissible to humans via direct contact or contaminated milk
  6. 6. Species Corynebacterium diphtheriae Laboratory diagnosis • Colection of specimens: throat / nasal / wound swab • Microscopy: – Gram positive bacilli, aspect of ”Chinese letters” / capital letters – low value (C.diphtheriae – similar to other comensal corynebacteria in the throat – “diphteroid bacilli”) • Cultivation: – Blood agar – Selective media with tellurite (Tinsdale, Gundel-Tietz) – Highly selective Loffler medium
  7. 7. Corynebacterium diphtheriae: Gram+, encurved rods, ”swolen” ends
  8. 8. Corynebacterium diphtheriae – Gram+, encurved rods, “swollen” ends
  9. 9. Corynebacterium diphtheriae - Cultivation - • Blood agar: nonhemolytic, white-grey colonies, striated margins (”daisy flower”)
  10. 10. Corynebacterium diphtheriae - Cultivation - continued • Tinsdale medium (tellurite): small, black colonies + brown surrounding area (reaction between H2S produced by bacteria and potasium tellurite in medium)
  11. 11. Corynebacterium diphteriae on Tinsdale agar: black colonies with brown halo
  12. 12. Left: Corynebacterium diphtheriae on blood agar Right: Corynebacterium diphtheriae on tellurite
  13. 13. Corynebacterium diphteriae - Cultivation - continued • Loffler medium – high selectivity: white, creamy colonies on slant
  14. 14. Biochemical tests: Urease test (medium: urea + phenol red indicator) • Principle: urease producing germs decompose urea in the culture medium: PINK • Corynebacterium diphteriae – NEGATIVE TEST – tube on the right • diphteroid bacilli (normal flora) – POSITIVE TEST – tube in the middle
  15. 15. Toxigenic vs Nontoxigenic strains - Toxigenesis tests - A. In vivo: experimental disease in Guinnea pigs B. In vitro: Elek´s test: • Elek agar inoculated with streaks of bacterial culture; • strip of sterile filter paper impregnated with antidiphteric antitoxin placed perpendicular to bacterial streaks; • incubate 24 hours; • POSITIVE test = lines of Ag- Ab precipitate = strain is toxigenic (important differentiation from diphteroids – normal flora of the pharynx)
  16. 16. Elek test - continued Elek agar: agar + bovine serum 1 and 4 – Elek (+) = double radial immunodiffusion – precipitation lines in each angle formed by bacterial streak and strip impregnated with diphteric antitoxin = toxin producing strains 2 and 3 = Elek (-) – nontoxigenic strains
  17. 17. Corynebacterium diphteriae Antimicrobial sensitivity • Sensitive to penicillin, vancomycin, erythromycin – given asap, based on clinical suspicion – before lab confirmation! • + treatment must include Diphteria ANTITOXIN !! • Lack of/ delayed treatment may cause death (effects of toxin – inhibition of protein synthesis; organ necrosis – e.g. heart, liver, kidneys; neurologic lesions) IMPORTANT: Diphteria – vaccine-preventable disease!! • Vaccination protocols e.g. – trivalent vaccines: (Diphteria, Tetanus, Pertusis); – pentavalent vaccines (Hemophilus influenzae type B, Pertusis, Tetanus, Diphteria, Hepatitis B)
  18. 18. Gram positive and Gram negative bacilli Genera: • Corynebacterium • Listeria • Erysipelothrix • Bacillus Family Enterobacteriaceae Genera: A. Highly pathogenic: • Yersinia • Salmonella • Shigella A. Facultatively pathogenic: • E.coli • Klebsiella • Proteus • Enterobacter • Serratia • Citrobacter
  19. 19. Genus Listeria Common characters: • Gram positive, short bacilli (rods) /cocobacilli, aerobic, non-spore forming, length: 0.5- 1 µm/diameter: 0.5 µm, rounded ends • Arrangement in palisades / V- shape / chains • Mobility due to peritrichous flagella (flagella around the rod) – up to 5
  20. 20. Genus Listeria - Clinical significance - • Natural habitat: soil, water, sewage, plants, foods (raw, undercooked) • Species involved in human pathology: Listeria monocytogenes, Listeria seeligeri, Listeria ivanovii • Transmission via contaminated food (milk, diary products, poultry meat) – food poisoning • Transplacentar transmission → meningitis, sepsis in newborns • Immunosuppressed patients (HIV, blood malignancies): meningitis, encephalitis, sepsis • Occupational infection in veterinarians, slaughterhouse workers (from infected animals/animal tissues): pulmonar infections
  21. 21. Genus Listeria - Laboratory diagnosis - • Collection of specimens: CSF, blood, amniotic fluid, respiratory secretions, food samples, etc. • Microscopic examination: difficult to differentiate from: – corynebacteria, – Hemophilus influenzae (excessive decoloration of smear), – streptococci (short rods/coccobacilli with rounded ends sometimes resemble ovoid cocci)
  22. 22. Listeria – Gram stained smear
  23. 23. Genus Listeria - Laboratory diagnosis – continued Cultivation: • Normally sterile collection sites (CSF, blood): – Inoculation in liquid media (enriched nutrient broth), 35°C, 5-7 days (daily examination for bacterial growth) – Reinoculation from turbid broth tubes on blood agar, incubation for 2 more days • Nonsterile collection sites (faeces, respiratory secretions, foods): – Initial inoculation on enrichment media – Reinoculation on selective media (e.g. with antibiotic content)
  24. 24. Genus Listeria - Laboratory diagnosis – continued Colonial characters: • Blood agar: small (up to 1 mm), round, smooth, translucent colonies, discrete hemolysis (sometimes under the colony, visible only after picking up the colony with loop) • Selective media (e.g. Oxford agar): black colonies, black halo (formation of iron compounds)
  25. 25. Listeria colonies on blood agar
  26. 26. Listeria monocytogenes on Oxford agar • Black colonies, black halo
  27. 27. Listeria: main bacteriological diagnosis elements
  28. 28. Listeria - Biochemical tests - • CAMP test – POSITIVE for L.monocytogenes • (enhanced hemolysis in the area where Listeria streak meets S.aureus streak) • CAMP test is negative for nonpathogenic Listeria strains
  29. 29. Gram positive and Gram negative bacilli Genera: • Corynebacterium • Listeria • Erysipelothrix • Bacillus Family Enterobacteriaceae Genera: A. Highly pathogenic: • Yersinia • Salmonella • Shigella A. Facultatively pathogenic: • E.coli • Klebsiella • Proteus • Enterobacter • Serratia • Citrobacter
  30. 30. Genus Erysipelothrix • Common characters: Gram positive bacilli, non spore forming, tendency to form long filaments • Clinical significance: zoonosis; human diseases occur as work-related infections (veterinarians, butchers, fishermen): inflammatory skin lesions (erysipeloid)
  31. 31. Gram positive and Gram negative bacilli Genera: • Corynebacterium • Listeria • Erysipelothrix • Bacillus Family Enterobacteriaceae Genera: A. Highly pathogenic: • Yersinia • Salmonella • Shigella A. Facultatively pathogenic: • E.coli • Klebsiella • Proteus • Enterobacter • Serratia • Citrobacter
  32. 32. Genus Bacillus Species Bacillus anthracis • Large, Gram positive rods (10 µM), • straight cut ends, • aerobic, • disposed in chains • Spore forming
  33. 33. Bacterial survival outside host Spores: reproductive structures adapted for longtime survival in unfavourable conditions (etymology: ancient Greek spora = seed) Bacterial spores - outer layer of keratin resistant to chemicals, staining and heat → bacterium able to stay dormant for years, protected from temperature differences, absence of air, water and nutrients Spore forming bacteria: • Clostridium spp (e.g. Clostridium difficile, Clostridium tetani); • Bacillus spp (B. anthracis).
  34. 34. Bacillus anthracis – Gram staining • Gram positive, long bacilli, squared ends, endo-spores visible in the middle of bacterial cells – spores are resistant to staining (magnification 1500X)
  35. 35. Bacillus anthracis (continued) • High pathogenicity • Disease = zoonosis (infection of animals AND humans) • Clinical forms: – Cutaneous anthrax – spores enter the body via skin lesions – Pulmonary anthrax – inhalatory infection – Digestive anthrax – ingestion of infected undercooked meat – + biological weapon (inhalatory infection) – agent of bioterrorism
  36. 36. Left: anthrax skin lesion Right: ”anthrax attack” letter (1 week after 9/11)
  37. 37. Bacillus anthracis: cultivation • Nonfastidious germ; grows well on blood agar • Colonial characters: large (2-5 mm) white colonies, non- hemolytic, irregular margins, sometimes comma shaped (lower image), ground-glass aspect (above details visible under magnifying glass)
  38. 38. Bacillus anthracis on blood agar

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