This document discusses Corynebacterium diphtheriae, the bacteria that causes diphtheria. It describes the morphology, culture properties, virulence factors, pathogenesis, epidemiology, laboratory diagnosis, prevention and treatment of diphtheria. Key points include that C. diphtheriae is a gram-positive rod that produces an exotoxin causing local inflammation and systemic effects. Laboratory diagnosis involves microscopy, culture and toxigenicity testing. Immunization with diphtheria toxoid vaccine provides strong lifelong immunity against the disease. Prompt antitoxin treatment is also important for therapy.

1. Corynebacteria. Causative agents
of diphtheria. Pathogenesis,
laboratory diagnostics, prophylaxis
and therapy of diphtheria.
Vinnitsa National Pirogov Memorial Medical University /
Department of microbiology

2. Taxonomy and classification of
corynebacteria
• Genus Corynebacterium
• Medical important species:
• C. diphtheria is a major human pathogenic
corynebacteria, causing diphtheria
• Conditionary-pathogenic species named
"diphtheroids" or "coryneform" habitat upper
respiratory tract and skin. This group comprises C.
ulcerans. C. haemolyticum, Cpseudodiphthericum.
C. xerosis and others

3. Morphological features
Corynebacteria are:
• Gram-positive, non-sporforming rods,
• sometimes club-shaped
• sometimes containing volutin granules,
• arranged in smears in angular or palisade clusters;
• non-motile,
• non-capsulated
• Non-acid-fast

4. Corynebacterium diphtheria
Morphological features
• They are slender rods with a tendency to clubbinq
at one or both ends, measuring approximately 3-6
pm x O.2-0.8pm.
• Granules (metachromatic or Babes Ernst granules)
composed of polymetaphosphate are stained with
Loeffler's methylene blue and with Neisser
technique. They are often situated at the poles of
the bacilli and are called polar bodies.

5. Morphological features
• The bacilli are arranged in pairs or small groups,
and being at various angles to each other bacilli
resemble
the letters V, W,X,Y or L
• This has been called the
Chinese letter or
cuneiform
arrangement.

6. C.diphtheriae
stained by Gram’s Stained by Neisser

7. Cultural properties
• C. diphtheria is an aerobe and facultative
anaerobe, has an optimum temperature for
growth of 37°C,
• It grows best on media containing blood or serum,
e.g. Loeffler's coagulated serum or blood agar
containing fresh, lysed or heated blood.
• It is isolated from clinical samples by culture on the
selective tellurite media which inhibit most other
pathogenic and commensal bacteria.

8. Cultural properties on the tellurite
media

9. Classification of C.diphtheria
• Based on colonial morphology on the tellurite
medium and other properties, McLeod classified
diphtheria bacilli into three types:
1. gravis,
2. intermedius
3. mitis.
• The names were originally proposed to relate to the
clinical severity of the disease produced by the three
types - gravis, causing the most serious, and mitis
the mildest variety, with intermedius being
responsible for disease of intermediate severity

10. Different biotypes of C.diphtheriae

11. Classification of C.diphtheria
• Gravis strains form large, grey-black (slate-
coloured), lustreless matt-surfaced colonies which
in old cultures may have a radial striate, 'daisy
head' appearance; they give a granular growth in
broth.
• Mitis strains form convex, grey-black colonies
with a matt but glistening surface, and give
uniformly turbid growth in broth.
• Intermedius strains form small grey-black
lustreless colonies with a 'poached egg'
appearance

12. Different types of colonies
Gravis variant Mitis variant

13. Biochemical differentiation of
Corynebacteria
Name of
species
Glucose
fermentatio
n
Sucrose
fermentatio
n
Starch
splitting
Urease Cystin
ase
C.diphtheria
v.gravis
+ - + - +
C.diphtheria
v.mitis
+ - - - +
C.diphtheria
v.intermedius
+ - - - +
C.xerosis
+ + - - -
C.pseudodipht
hericum
- - - + -
C.ulcerans
+ + + + +
JK group
+ +/- - - -

14. Resistant:
• Cultures may remain viable for two or more
weeks at 25-30 °C
• It is relatively high resistant to UVR,
desiccation and freezing.
• It survives into the dried bits of
pseudomem-brane after 14 weeks and into
the floor dust for 5 weeks.
Sensitivity:
• It is readily destroyed by heat in 10 minutes
at 58 °C and in a minute at 100 °C
• It is easily destroyed by antiseptics.

15. Virulent factors
1. Exotoxin (cytotoxin).
• It inhibits protein synthesis : inhibition of
polypeptide chain elongation by inactivating
the elongation factor, EF-2. It has a special
affinity for certain tissues such as the
myocardium, adrenals and nerve endings
2. Enzymes: fibrinolysin, neuramidase, hyaluronidase.
• Responsible for typical inflammation into the entry of
diphtheria bacilli (pseudomembranous on the flat
multilayer epithelium and membranous on the
monolayer epithelium)

16. Epidemiology
• The source of infection is either a ill person
or a carrier
• Infection is spread by droplets, dust,
secretions, or direct contact.
• It is air-borne infection (nasopharyngeal
diphtheria).
• Cutaneous diphtheria is transmitted with
direct contact
• High risk group: children up to 5 y.o.
• The incubation period in diphtheria is
commonly 3—4 days.

17. Pathogenesis
Site of entry: multiplication of
bacilli and formation of the
cytotoxin
Local inflammation due to enzymes and
toxin action: diphtheric pseudomembrane
on flat multilayered epithelium and crupous
membrane on monolayered epithelium
Toxemia and systemic action on
target cells (heart, adrenals and
peripheral nerves)

18. Pathogenesis
• Diphtheria is a toxemia.
• The toxin causes local necrotic changes and the
resulting fibrinous exudate, together with the
disintegrating epithelial cells, leucocytes,
erythrocytes and bacteria, constitute the
pseudomembrane, which is characteristic of
diphtheritic infection.
• Complications: The mechanical complications
(croup) of diphtheria are due to the membrane,
while the systemic effects are due to the toxin.

19. Clinical appearance
Membrane onto the tonsils

20. Clinical appearance
Nasal bleeding Edema

21. Immunity
• It is antibacterial and antitoxic after disease and
antitoxic after immunization.
• Immunity is strong, it lasts for some decades or
even whole life.

22. Laboratory diagnostics
1. Microscopy (presumptive diagnosis) The smears
are stained by Neisser or Loeffler
2. Culture method and determination of
toxigenecity (confirming diagnosis)
3. Immunofluorescence microscopy (rapid
diagnostics)
4. Experimental infection for toxigenecity detection
Collected samples: swab smear from the lesions,
exudates, membranes

23. Microscopy

24. Culture method
• For culture, the swabs are inoculated and tellurite
blood agar or Tinsdale's medium
• After overnight incubation suspected colonies are
subcultured on Loeftier's serum slope agar
• After 6-8 hours subculture is identified with
biochemical testing and toxigenecity testing

26. Toxigenecity testing
• Any isolate of the diphtheria bacillus must be
tested for virulence or toxigenicity for the
bacteriological diagnosis to be complete.
• Virulence testing may be by:
1. in vivo (subcutaneous or intradermal test in
guinea pigs) methods
2. or in vitro methods ( precipitation test in a gel or
the tissue culture test)

27. Subcutaneous test
• The growth from an overnight culture on Loeffler's
slope is emulsified in broth and the emulsion is
injected subcutaneously into two guinea pigs, one
of which has been protected with diphtheria
antitoxin 18—24 hours previously.

28. Subcutaneous test
• If the strain is virulent, the unprotected animal will
die within 4 days.
At autopsy, the following features can be observed:
• gelatinous, hemorrhagic edema and, often,
necrosis at the site of inoculation,
• swollen and congested draining lymph nodes,
• peritoneal exudate
• congested abdominal viscera, enlarged
hemorrhagic adrenals, which is the
pathognomonic feature

29. In vitro test
Elek's gel precipitation test:
A strip of filter paper impregnated with the
diphtheria antitoxin is placed on the surface of a
20% normal horse serum agar. Narrow streaks of
the strains are made at right angles to the filter
paper strip. A positive and negative control should
be put up.
The plate is incubated at 3f°C for 24-48 hours. Toxins
produced by the bacterial growth will diffuse in the
agar and where it meets the antitoxin at optimum
concentration will produce a line of precipitation

30. Elek`s gel precipitation test

31. Prophylaxis
• Diphtheria can be controlled by immunization.
• Three methods of immunisation are available:
active, passive and combined.

33. Active prophylaxis
• Diphtheria toxoid (DT) is usually given in children
as a trivalent preparation containing tetanus
toxoid and pertussis vaccine also, as the DPT
• The schedule of primary immunization of infants
and children consists of three doses of DPT given
at intervals of at least four weeks, followed by a
fourth dose about a year afterwards.
• A further booster dose is given at 6, 11 and 14-15
years old (DTT).

34. Therapy of diphtheria
• Antitoxic therapy should be started immediately
(!).
• The dose of antitoxic horse serum depends on the
severity disease and is from 20 000 to 100 000 IU
• Antibiotics
(penicillin, erythromycin)

35. Corynebacterium diphtheriae

36. Bordetella
Family: Brucellaceae
Genus: Bordetella
Pathogenic species:
B.pertussis causes whooping cough (pertussis)
B.parapertussis causes parapertussis
B.bronchiseptica causes bronchosepticosis

37. Morphology
• Gram-negative short,
coccoid, nonmotile
rods
• Non-sporeforming
• B.pertussis forms
microcapsule

38. Cultivation
• Obligate aerobes
• Optimal temperature 35-370C
• Can not grow on the ordinary media due to
self-inhibition with metabolic products (fatty
acids)
• Cultivated on the complex media with sorbent
compounds, enriched with natural proteins
(blood, casein, plasma proteins, coal and
others)
• Slow growth (within 3-5 days for B.pertussis,
and 2 days for other Bordetella)

39. Culture media and growth appearance
Special media for Bordetella cultivation
1.Bordet-Gengou medium (glycerol-potato-
blood agar)
2. Charcoal-blood agar
3. Charcoal-casein agar
Colonies are small, opaque, smooth, greyish-
white, refractive (“bisected pearls” or
“mercury drops” appearance)

40. Growth on the charcoal-blood agar

41. Antigenic structure and serotyping
of Bordetella
K- antigen is heat labile, designated 1 to 14
factors revealed with agglutination test
Factor 7 of K-ag is found in all pathogenic
Bordetella (B.pertussis,B.parapertussis, and
B.bronchoseptica)
Species specific factors:
Factors1-6 : B.pertussis (the most often 1,2,3)
Factor 12 : B.parapertussis
Factor 14: B.bronchoseptica

42. Virulent factors
• Heat stable LPS of the cell wall
• Action: general intoxication, fever
Endotoxin
• Filamentous haemagglutinin (FHA)
• Adhesion to ciliated epithelial cells
Pili
• Neuraminidase
• Plasmocoagulase
• Adenylate cyclase (AC)
Enzymes

43. Pertussis toxins
• Heat labile toxin, cytoplasmic
protein
HLT
• Tracheal cytotoxin, stimulate cytokines
production, causing death of tracheal
epithelium
• Derived from peptidoglycane, heat stable
TCT
• Pertussis toxin, heat labile
• Lymphocytosis producing factor, histamine
sensitising factor, islet activating factor
PT

44. Epidemiology
The source of infection is ill person (during
catarrhal stage)
Mechanism of transmission by inhalation .
It is air-borne infection transmitted via droplets.
High risk group: children, especially dangerous
for 1st year children (bronchopneumonia).
Mostly whooping cough is caused by B.pertussis
(90-95%), other cases: B.parapertussis (5-10%),
and few ones – B.bronchoseptica

45. Pathogenesis
Incubation period: 1-2 weeks
Duration of disease:
1. Catarrhal stage (most contagious) takes
about 2 weeks
2. Paroxysmal stage (spastic violent cough)
– 2 weeks
3. Convalescent stage (recovering period
with less coughing) – 2 – 4 weeks
Immunity: strong, life-lasting, antibacterial
and antitoxic

46. Typical violent coughing

47. Laboratory diagnostics
Main methods:
1. Bacteriological (pure culture isolation)
Reliable during catarrhal stage
Collection of samples with nasopharyngeal
swabs; “cough plates”
Additional method during this stage:
immunofluorescent microscopy
2. Serology (after 2nd week of disease)
Detection of patient’s antibody in the collected
blood with agglutination test, complement
fixation test

48. Bacteriological method

49. Prevention and treatment
Time table vaccination with DPT vaccine (3-6 months
three times), followed by booster dose in a year
Vaccines (as a part of associated vaccines):
1. Inactivated cellular vaccine (B.pertussis, phase 1)
as a part of DPT
2. Chemical vaccine containing pertussis toxoid
(PT), filamentous hemagglutinin (FHA) and K-Ag
(factors 1,2,3) as a part of modern associated
vaccines (Infanrix, Pentaxim, others)
Contact persons (current prevention) at age before 5
years: booster dose of vaccine and erythromycin