1. Corynebacteria

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

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.

4. Corynebacterium spp
Gram positive bacilli, with characteristic morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Non--hemolytic on sheep blood agar
Facultative anaerobic
C. diphtheriae is fastidious while diphtheriods are non-fastidious
Catalase positive
Oxidase negative

5. ØLipid-rich cell wall contains meso-diaminopimelic acid, arabino-galactan polymers, and short-chain mycolic acids
ØLysogenic bacteriophage encodes for potent exotoxin in virulent strains

6. Species of Corynebacteria
Corynebacterium diphtheriae
Other Significant Corynebacterium species
C. xerosis
C. pseudodiphtheriticum
C. pseudotuberculosis
C. jeikeium, (skin)
C. ulcerans
Normal flora of RT, urethra, vagina, Skin

7. Introduction – C. diphtheriae
Diphtheros – leather (tough, leathery pseudomembrane)
Also known as Klebs–Loeffler bacillus
Causes Diphtheria

8. 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

11. 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

12. 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

14. Mechanism of Action of Diphtheria Toxin:
Mechanism of Action of Diphtheria Toxin: Inhibition of Protein Synthesis

15. Molecular Structure of Diphtheria Toxin
Catalytic Region
Receptor-Binding Region
Translocation Region
A Subunit
B Subunit

16. 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.
Antibodies to fragment B - protective

17. 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

18. 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

19. 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)

21. 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.

22. 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

24. Diphtheria Cased by C. diphtheriae Mycocarditis, neuroitis,
Acute, Toxin mediated
Recovery or complication & death (if more toxin absorbed)
Childhood disease affect upper respiratory tract
Diphtheria
Respiratory obstruction due to extensive membrane formation
Transmitted by droplet infection
2-6 days I.P.
Sore throat, Pharyngitis
2-3 days, Bluish white adherent pseudo membrane

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

26. Morphology Gram +ve, nonspore forming nonmotile bacilli
Club-shaped (Coryne= club) arranged at acute angles or parallel to each other (Chinese letters appearance)
Beaded (metachromatic granules)
Gram stain:
C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped

27. Biochemical Reaction
All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

28. 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

29. Growth of diphtheria bacilli
Blood agar
Tellurite blood agar
Loeffler’s serum slope

30. 3 biotypes of C. diphtheriae are characterized on BTA
i.e. Gravis, mitis and intermedius biotypes
The most severe disease is associated with the gravis biotype
Colony of gravis biotype is large, non-hemolytic & grey.
Colonies of mitis biotype are small, hemolytic and black
Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

31. 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

32. Laboratory Diagnosis Biochemical reactions
Ferments sugar with acid formation but not Gas ferments: glucose, galactose, maltose and dextrin, but not ferment sucrose.
Resistant to light, desiccation and freezing
Sterilization: sensitive to heat (destroyed in 10mins at 58°C or 1min in 100°C), chemical disinfectants

33. 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

34. 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

35. In vivo Detection of Diphtheria Exotoxin

36. 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

37. Detection of toxin: Elek’s Test
Principle:
It is toxin/antitoxin reaction
Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin
Procedure:
A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar
The organism is streaked at right angels to the filter paper
Incubate the plate at 37C for 24 hrs

38. Filter paper saturated with diphtheria antitoxin
Resuls:
After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism
Lines of precipitations
Inoculated M.O.
Positive Elek’s Test

39. 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

40. Toxin - Antitoxin Neutralization test
In Vivo (Schick test):
Schick test - used to determine whether or not a person is susceptible to diphtheria (Corynebacterium diphtheriae).
Named after its inventor, Béla Schick ( )
It is a simple procedure. 40

41. A small amount (0.1 ml) of diluted (1/50 MLD) diphtheria toxin is injected intradermally into the arm of the person.
Minimum lethal dose (MLD, also LDmin) is the least amount of drug that can produce death in a given animal species under controlled conditions
If a person does not have enough antibodies to fight it off, the skin around the injection will become red and swollen, indicating a positive result.
This swelling disappears after a few days.
If the person has immunity, then little or no swelling and redness will occur, indicating a negative result. 41

42. Results can be interpreted as:
Positive: when the test results in a red necrotic area of 5-10 mm diameter
Pseudo-positive: when there is only a red colored inflammation and it disappears rapidly
Negative reaction: No Wheel and erythema

43. 43

44. 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 44

45. 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) 45

46. 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

47. 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

48. CONTROL isolate patients treat with antibiotics actively
complete vaccination schedule should be used with booster every 5 years

49. 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