1. Gram positive rods
Dr Alex Owusu-Ofori

2. Gram positive rods Non spore-forming AEROBIC ANAEROBIC Spore-forming
Corynebacteria
C. diphtheriae
diphtheroids
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

3. Species of Corynebacteria
Corynebacterium
Pathogenic
C. diphtheriae
Commensal "Diphtheriods"
C. hofmannii, C. xerosis, C. acne
C. diphtheriae is the only pathogenic members of this genus
Normal flora of RT, urethra, vagina, Skin

4. Corynebacteria - Overview
Gram positive, non motile bacilli with irregularly stained segments
C. diphtheriae : most important member of this genus, causes diphtheria
Diphtheroids : commensals of nose, throat, nasopharynx, skin, urinary tract & conjunctiva.
Corynebacteria is the most successfully studied bacteria – clinical description started by Hippocrates in the 4th Century BC

5. Historical overview I. Corynebacterium diphtheriae (Klebs–Loeffler bacillus)
Bretonneau 1826
Clinical characterisation
of diphtheria – diphtherite
Klebs 1883
First decribed the bacterium
Loeffler 1884
First solated/cultivated 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

6. Historical overview II.
Emil von Behring 1901
Produced antitoxin - 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)

7. Corynebacterium spp
Gram positive bacilli, with characteristic morphology (club shaped and beaded)
Non motile
Non spore forming
Non capsulated
Facultative anaerobic (A facultative anaerobe is a microorganism that can exist regardless of whether there is molecular oxygen. When there is oxygen, the anaerobe creates ATP using aerobic respiration. If oxygen is not present, then the organism uses anaerobic resp or fermentation)
Breakdown glucose by oxidation and fermentation
C. diphtheriae is fastidious while diphtheriods are non-fastidious
Catalase positive
Oxidase negative

8. Important features of C. diphtheriae
Slender Gram positive bacilli
Pleomorphic,
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

9. 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

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.
Strain used for toxin production – ‘Park Williams 8’ strain
Antibodies to fragment B - protective

11. Epidemiology
Habitat – nose, throat, nasopharynx & skin of carriers and patients
Spread by respiratory droplets, usually by convalescent or asymptomatic carriers
Nasal carriers habour 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

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

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)

14. 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

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.

16. Diagnosis of diphtheria
Clinical Diagnosis
Laboratory Diagnosis
Specific treatment must be never delayed for laboratory results
To confirm the clinical manifestation

17. Diagnosis of diphtheria
Diagnosis of case
Diagnosis of carrier
Symptomatic patient
Asymptomatic

18. Laboratory diagnosis Specimen: A throat swap
Culture: The swap is inoculated on Loeffler's serum medium and/or on blood tellurite agar aerobically at 37°C for 24.
On Loeffler's serum medium:
Corynebacteria grow much more readily than other respiratory pathogens
Used to enhance the characteristic microscopical appearance of corynebacteria
The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges

19. Loefflers serum medium
Developed by Friedrich Loeffler in 1887
Loefflers medium is used to grow corynebacteria
Most recent modification by Buck in 1940 to enhance the growth of C. diphtheria
Consists of Beef serum, heart muscle and animal tissue peptone, sodium chloride, dextrose and egg.
The primary value of Loeffler Medium is in the growth and morphological characterization of members of the genus Corynebacterium; enhances the formation of metachromatic granules within the cells of the organism.

20. 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

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

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

23. Gram stain of C. diphtheriae
C. diphtheriae on BTA

24. Cultural characteristics
On blood tellurite agar (Mcloed’s blood agar)
It is selective medium for isolation of C. diphtheriae (Potassium tellurite)
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.

25. Morphology Stain 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)
Stain
Gram stain:
C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped
Polychrome methylene blue stain:
C. diphteriae appears beaded due to the presence of intercellular “Metachromatic or volutin" granules
By stain, the granules appear red while the rest of organism appears blue.

26. 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

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

28. Carbohydrate Fermentation Test:
Principle:
Each species of corynebacteria has its specific carbohydrate fermentation pattern
C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only
Procedure:
Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism
Glucose
Maltose
Sucrose
Incubate the tubes at 37oC for 24 hrs.

29. Results:
Sugar fermentation can be indicated by change of color of the medium from red to yellow due to formation of acid which decrease the pH
Glucose
Maltose
Sucrose
Glucose
Maltose
Sucrose
+ve
+ve
+ve
+ve
+ve
- ve
C. xerosis
C. diphtheriae

30. 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
Dr Ekta, Microbiology

31. 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

32. 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)

33. 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

34. 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

35. Diphtheria Cased by C. diphtheriae
Mycocarditis, neuroitis, palate perforation
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