Corynebacteria

1. Corynebacteria II MBBS Dr Ekta Chourasia Microbiology

2. Non spore-forming AEROBIC Corynebacteria C. diphtheriae diphtheroids C. jeikeium Listeria monocytogenes Erysipelothrix rusiopathiae ANAEROBIC Lactobacillus spp. Spore-forming AEROBIC Genus: Bacillus B. anthracis B. cereus B. subtilis ANAEROBIC Genus: Clostridium C. tetani C. botulinum C. difficile C. perfringens Gram positive rods Dr Ekta, Microbiology

3. Corynebacteria - Overview • Gram positive, non motile bacilli with irregularly stained segments • Frequently show club shaped swellings – corynebacteria (coryne = club) • C. diphtheriae : most important member of this genus, causes diphtheria • Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva. Dr Ekta, Microbiology

4. Bretonneau 1826 Clinical characterisation of diphtheria – diphtherite Klebs 1883 Detecting the bacterium Loeffler 1884 Isolating the bacterium Roux and Yersin 1888 Discovering the diphtheria toxin Behring and Kitasato 1890-1892 Discovering the diphtheria antitoxin Antitoxic immunity (therapy and prevention) Roux 1894 Treatment with antitoxin Historical overview I.Corynebacterium diphtheriae Dr Ekta, Microbiology

5. Emil von Behring 1901 Nobel prize Behring 1913 Active immunisation I. with toxin-antitoxin mix Schick 1913 Skin test Ramon 1923 Active immunisation II. Anatoxin = toxoid Freeman 1951 PHAGE (lysogenia, toxin production) Historical overview I.Corynebacterium diphtheriae Dr Ekta, Microbiology

6. Introduction – C. diphtheriae • Diphtheros – leather (tough, leathery pseudomembrane) • Also known as Klebs–Loeffler bacillus • Causes Diphtheria Dr Ekta, Microbiology

7. Important features of C. diphtheriae • Slender Gram positive bacilli • Pleomorphic, non motile, non sporing • Chinese letter or Cuneiform arrangement • Stains irregularly, tends to get easily decolorised • May show clubbing at one or both ends - Polar bodies/ Metachromatic granules/ volutin or Babes Ernst granules • Metachromatic Granules: • made up of polymetaphosphate • Bluish purple color with Loeffler’s Methylene blue • Special stains: Albert’s, Neisser’s & Ponder’s • Grows aerobically at 37°C Dr Ekta, Microbiology

8. Virulence factor • Exotoxin – Diphtheria toxin: • Protein in nature • very powerful toxin • Responsible for all pathogenic effects of the bacilli • Produced by all the virulent strains • Two fragments A & B Dr Ekta, Microbiology

9. Diphtheria toxin – Mechanism of action DT - Acts by inhibition of protein synthesis Fragment A – inhibits polypeptide chain elongation by inactivating the Elongation factor EF 2 in the presence of NAD Dr Ekta, Microbiology

10. Diphtheria Toxin • Toxigenicity can be induced by Lysogenic or phage conversion – corynephages (tox+ phage) or beta phages • Can be toxoided by - • Prolonged storage • Incubation at 37°C for 4 - 6 weeks • Treatment with 0.2 – 0.4 % formalin or • Acid pH. • Stain used for toxin production – ‘Park Williams 8’ strain • Antibodies to fragment B - protective Dr Ekta, Microbiology

11. Epidemiology • Habitat – nose, throat, nasopharynx & skin of carriers and patients • Spread by respiratory droplets, usually by convalescent or asymptomatic carriers • Nasal carriers harbour the bacilli for longer time than pharyngeal carriers • Local infection of throat - toxemia • Incubation period of diphtheria – 3 to 4 days • In tropics, cutaneous infection is more common than respiratory infection Dr Ekta, Microbiology

12. Diphtheria • Site of infection • Faucial (palatine tonsil) – commonest type • Laryngeal • Nasal • Otitic • Conjunctival • Genital – vulval, vaginal, prepucial • Cutaneous – usually a secondary infection on pre-existing lesion, caused by non toxigenic strains Dr Ekta, Microbiology

13. Pathogenesis & Clinical Manifestations • Human Disease • Usually begins in respiratory tract • Virulent diphtheria bacilli lodge in throat of susceptible individual • Multiply in superficial layers of mucous membrane • Elaborate toxin which causes necrosis of neighboring tissue cells • Inflammatory response eventually results in pseudomembrane(fibrinous exudate with disintegrating epithelial cells, leucocytes, erythrocytes & bacteria) • Usually appears first on tonsils or posterior pharynx and spreads upward or down • In laryngeal diphtheria, mechanical obstruction may cause suffocation • Regional lymphnodes in neck often enlarged (bull neck) Dr Ekta, Microbiology

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15. Diphtheria - Clinical Classification • Based on the severity of clinical presentation: • Malignant or hypertoxic – severe toxemia with marked adenitis • Septic – ulceration, cellulitis, & gangrene around the pseudomembrane • Hemorrhagic – bleeding from the edge of membrane, epistaxis, conjunctival hemorrahge, purpura & generalized bleeding tendency. Dr Ekta, Microbiology

16. Complications of diphtheria • Mechanical complications are due to the pseudomembrane, while the systemic effects are due to the toxin. • Asphyxia – due to obstruction of respiratory passage • Acute circulatory failure • Postdiphtheritic paralysis – occurs in 3rd or 4th week of disease, palatine & ciliary, spontaneous recovery • Sepsis – pneumonia & otitis media Dr Ekta, Microbiology

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18. Laboratory Diagnosis • Specimen – swab from the lesions • Microscopy • Gram stain: Gram +ve bacilli, chinese letter pattern • Immunofluorescence • Albert’s stain for metachromatic granules Dr Ekta, Microbiology

19. Laboratory Diagnosis • Culture – isolation of bacilli requires media enriched with blood, serum or egg • Blood agar • Loeffler’s serum slope – rapid growth, 6 to 8 hrs • Tellurite blood agar – tellurite is reduced to tellurium, gives gray or black color to the colonies • Hoyle’s media modifications of TBA • McLeod’s media Dr Ekta, Microbiology

20. Growth of diphtheria bacilli Blood agar Tellurite blood agar Loeffler’s serum slope Dr Ekta, Microbiology

21. Biotypes of Diphtheria bacilli • Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types: Dr Ekta, Microbiology

22. Laboratory Diagnosis • Biochemical reactions • Hiss's serum water- ferments sugar with acid formation but not Gasferments: glucose, galactose, maltose and dextrin • Resistant to light, desiccation and freezing • Sterilization: sensitive to heat (destroyed in 10mins at 58°C or 1min in 100°C), chemical disinfectants Dr Ekta, Microbiology

23. Laboratory Diagnosis • Virulence tests - Test for toxigenicity • Invivo tests – animal inoculation (guinea pigs) • Subcutaneous test • Intracutaneous test • Invitro tests • Elek’s gel precipitation test • Tissue culture test Dr Ekta, Microbiology

24. Laboratory DiagnosisVirulence tests - Invivo tests • Bacterial growth from Loeffler’s serum slope is emulsified in 2-4 ml broth. • Two guinea pigs (GP A and GP B) • Subcutaneous test – 0.1 ml of emulsion is injected SC into each guinea pig GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B - Doesn't have antitoxin • Intracutaneous test - 0.1 ml of emulsion is injected IC into each guinea pig GP A - has diphtheria antitoxin (500 units injected 18 to 24 hours before) GP B – 50 units of antitoxin IP four hrs after the skin test Dr Ekta, Microbiology

25. Laboratory DiagnosisVirulence tests - Invitro tests • Elek's gel precipitation test • filter paper saturated with antitoxin (1000units/ ml) is placed on agar plate with 20% horse serum • bacterial culture streaked at right angles to filter paper Dr Ekta, Microbiology

26. Laboratory DiagnosisVirulence tests - Invitro tests • Tissue culture test- incorporation of bacteria into agar overlay of eukaryotic cell culture monolayers. Result: toxin diffuses into cells and kills them Dr Ekta, Microbiology

27. Treatment • specific treatment must not be delayed if clinical picture suggests of diphtheria • rapid suppression of toxin-producing bacteria with antimicrobial drugs (penicillin or erythromycin) • early administration of antitoxin: 20,000 to 1,00,000 units for serious cases, half the dose being given IV Dr Ekta, Microbiology

28. Prophylaxis • Active Immunization (Vaccination) • Formol toxoid (fluid toxoid) • incubation of toxin with 0.3% formalin at pH 7.4 - 7.6 at 37°C for 3 to 4 weeks • fluid toxoid is purified and standardized in flocculating units (Lf doses) • Adsorbed toxoid (more immunogenic than fluid toxoid) • purified toxoid adsorbed onto insoluble aluminium phosphate or aluminium hydroxide • given IM (DTP or TD) Dr Ekta, Microbiology

29. Prophylaxis • Adsorbed Toxoid • DPT - triple vaccine given to children; contains diphtheria toxoid, Tetanus toxoid and pertussis vaccine • DaT - contains absorbed tetanus and ten-fold smaller dose of diphtheria toxoid. (smaller dose used to diminish likelihood of adverse reactions) • Schedulei) Primary immunization - infants and children - 3 doses, 4-6 weeks interval - 4th dose after a year - booster at school entry ii) Booster immunization - adults -Td toxoids used (travelling adults may need more) • SHICK test - to test susceptibility to vaccine, not done now-a-days Dr Ekta, Microbiology

30. Prophylaxis • Passive immunizationADS (Antidiphtheritic serum, antitoxin) - made from horse serum - 500 to1000 units subcutaneously • Combined immunizationFirst dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation Dr Ekta, Microbiology

31. CONTROL • isolate patients • treat with antibiotics actively • complete vaccination schedule should be used with booster every 5 years Dr Ekta, Microbiology

32. Other Corynebacteria • C. ulcerans – diphtheria like lesions in guinea pigs & cows, may get transmitted to humans by cow’s milk • Diphtheroids – • Normal commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva • Stain uniformly • Few or no metachromatic granules • Arranged in parallel rows (palisades) • Nontoxigenic Dr Ekta, Microbiology