1. II MBBS Dr Ekta Chourasia Microbiology
Corynebacteria
II MBBS
Dr Ekta Chourasia
Microbiology

2. Gram positive rods Non spore-forming AEROBIC ANAEROBIC Spore-forming
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
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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.
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4. Historical overview I. Corynebacterium diphtheriae
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
Discovering the diphtheria antitoxin
Antitoxic immunity (therapy and prevention)
Roux 1894
Treatment with antitoxin
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5. Historical overview I. Corynebacterium diphtheriae
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)
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6. Introduction – C. diphtheriae
Diphtheros – leather (tough, leathery pseudomembrane)
Also known as Klebs–Loeffler bacillus
Causes Diphtheria
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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
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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
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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
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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 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
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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
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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
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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)
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14.
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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.
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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
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17.
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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
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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
BA – to differentiate from staphylococal or streptococcal pharyngitis
C.diph colonies are Small, granular, irregular edges and gray with small zones of hemolysis
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20. Growth of diphtheria bacilli
Blood agar
Tellurite blood agar
Loeffler’s serum slope
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21. Biotypes of Diphtheria bacilli
Based on colony morphology on the tellurite medium & other properties, McLeod classified diphtheria bacilli into three types:
Features
1. Gravis
2. Intermedius
3. Mitis
Case fatality rate
High
Low
Complications
Paralytic, hemorrhagic
Hemorrhagic
Obstructive
Predominance
In epidemic areas
Epidemic areas
Endemic areas
Spread
Rapid
Rapidly than mitis
Less rapid
Colony on TBA
‘Daisy head” colony
‘Frog’s egg colony
‘Poached egg’ colony
Hemolysis
Variable
Nonhemolytic
Usually hemolytic
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22. Laboratory Diagnosis Biochemical reactions
Hiss's serum water - ferments sugar with acid formation but not Gas ferments: 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
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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
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24. Laboratory Diagnosis Virulence 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 ml of emulsion is injected IC into each guinea pig
GP B – 50 units of antitoxin IP four hrs after the skin test
SC test – death within 4 days in GP B,
IC test – no death in any case, GP B – inflammatory reaction at the site of injection, progressing to necrosis in hrs
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25. Laboratory Diagnosis Virulence 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
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26. Laboratory Diagnosis Virulence 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
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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
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28. Prophylaxis Active Immunization (Vaccination)
Formol toxoid (fluid toxoid)
incubation of toxin with 0.3% formalin at pH 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)
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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)
Schedule i) 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
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30. Prophylaxis
Passive immunization ADS (Antidiphtheritic serum, antitoxin) - made from horse serum
- 500 to1000 units subcutaneously
Combined immunization First dose of adsorbed toxoid + ADS, to be continued by the full course of active immunisation
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31. CONTROL isolate patients treat with antibiotics actively
complete vaccination schedule should be used with booster every 5 years
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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
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