Corynebacterium

 Corynebacteria are gram positive , non- acid

fast , nonmotile rods with irregularly stained
segments , and granules.
 The most important member of this genus is
C. diphtheriae , the causative agent of
diphtheria.
 Klebs--1883 discovered and Loefflers--1884
cultured so also known as KLB or Klebs –
Loefflers bacillus.
 C. diphtheriae
 Morphology:
 Slender rods with a tendency to clubbing at
one or both ends.
 Nonmotile, noncapsulated, nonsporing
 Pleomorphic- shows branching infrequently.
 Gram positive bacilli but tends to
decolourise easily.
 Granules composed of polymetaphosphate
are seen in the cells.
 Stained with Loeffler’s methylene blue the
granules are seen in bluish-purple colour and
hence called metachromatic granules.
 Also called as volutin granules or babes ernst
granules.
 Special staining- Albert’s, Neisser’s & Ponder’s –
demonstrating granules.
 Bacilli- in smears- a characteristic
appearance- Usually seen in pairs,
palisades or small groups, bacilli being at
various angles to each other- resembling
letters of V or L. This has been called as the
Chinese letter pattern or cuneiform
arrangement. This is due to the incomplete
separation of daughter cells after binary
fission.
 Cultural characteristics:
 Growth is scanty on ordinary media.
 Enrichment with blood, serum or egg is
necessary for good growth.
 The optimum temperature for growth is 37⁰c
and the optimum pH is 7.2.
 It is an aerobe and a facultative anaerobe.
 The usual media employed for the cultivation of
the diphtheria bacillus are Loeffler's serum
slope and tellurite blood agar.
 Loeffler's serum slope
 Diphtheria bacilli grow on Loeffler's serum
slope very rapidly and clolonies can be seen
in 6-8 hours.
 Colonies are at first small, circular white
opaque discs but enlarge on continued
incubation and may acquire a distinct
yellow tint.
 Tellurite blood agar
 Modifications of tellurite blood agar used-
McLeod’s and Hoyle’s media.
 Tellurite inhibits the growth of other bacteria-
act as a selective agent. Diphtheria reduce
tellurite to metallic tellurium – colonies grey or
black colour.
 Growth of bacteria in tellurite medium is
delayed – takes 2 days to appear.
 Based on colony morphology on Tellurite medium
and other properties- McLeod classified
diphtheria bacilli into 3 types- gravis, intermedius
and mitis.
 Names originally related to the clinical severity of
the disease.
 Gravis – most serious. Intermedius- intermediate
severity and mitis – mildest type.
 Biochemical reactions:
 Ferment glucose, galactose, maltose and dextrin
with acid but no gas. Hiss’s serum water used for
testing sugar fermentation.
 Urease, phosphatase test - negative.
 Toxin:
 Virulent strains of diphtheria bacilli produce a
very powerful exotoxin. The pathogenic effect of
the bacilli are due to toxin.
Properties:
 Diphtheria toxin is a protein. It has two fragments, A
and B.
 Both the fragments are necessary for toxic effect.
 All the enzymatic activity of the toxin is present in
fragment A. Fragment B is responsible for the
binding the toxin to the cell
 Toxin is labile.
 Prolonged storage, incubation at 37ᵒ C for 4 – 6
weeks, treatment with 0.2-0.4 % formalin or
acid pH converts it to toxoid.
 Toxoid – toxin which has lost its toxigenicity but
not its antigenicity.
 Capable of inducing antitoxin.
 Toxin production coded by a phage –
corynephage(tox+).
 Mechanism of action
 Diphtheria toxin acts by inhibiting protein
synthesis.
 Fragment A inhibits polypeptide chain
elongation.
 This toxin have special affinity for certain
tissues such as myocardium , adrenals and
nerve endings.
 Pathogenicity:
 Incubation period is commonly 3-4 days.
 Site of infection may be:
– Faucial
– Laryngeal
– Nasal
– Otitic
– Conjunctival
– Genital
– Cutaneous – occurs as secondary infection
on existing skin lesions. Caused by
nontoxigenic strains.
 Faucial diphtheria is the commonest type and
may vary from mild catarrhal inflammation to
very wide spread involvement.
 According to clinical severity, diphtheria may
be classified as:
1. Malignant (hypertoxic) diphtheria-
Severe toxemia with marked
adenitis(lymph glands swelling in the neck).
Death- due to circulatory failure. Paralytic
sequelae occurs.
2. Septic diphtheria –
Leads to ulceration, cellulites and gangrene
around pseudomembrane.
 3. Hemorrhagic diphtheria –
Bleeding from edge of pseudomembrane,
conjunctival hemorrhage, epistaxis purpura
and generalised bleeding tendency.
 Common complications are:
 1)Asphyxia-obstruction of respiratory tract by
the pseudomembrane for which an
emergency tracheostomy may be necessary.
 2)Acute circulatory failure
 3)Post diphtheritic paralysis-soft palate, eye
muscles, extremities in the 3rd-4th week of
the disease.
 4)Septic sequelae- pneumonia, otitis media.
 5). Toxemia - Diphtheria is a toxemia. Bacteria
remain confined to the site of entry, there they
multiply and form toxins.
 Toxin causes local necrotic changes and the
resulting fibrinous exudate together with the
disintegrating epithelial cells, leucocytes,
erythrocytes and bacteria – constitute the
pseudomembrane- characteristic feature of
diphtheritic infections.
 Mechanical complications are due to membrane
and systemic effects are due to toxin.
 Laboratory Diagnosis:
 Necessary for the initiation of control measures
and for epidemiological purposes. Not for
treatment. Specific treatment should be given
immediately on suspicion of diphtheria without
waiting for laboratory tests.
 Consists of isolation of the diphtheria bacilli and
demonstration of its toxicity.
 Samples – two swabs from the lesion are collected
under vision – using tongue depressor.
 Microscopy – perform gram stain and albert
staining – not much reliable due to commensal
corynebacteria.
 Toxigenic diphtheria bacilli may be identified in
smears by immunofluorescence.
 Culture isolation- swabs are inoculated on
loeffler’s serum slope, tellurite blood agar and a
plate of ordinary blood agar( to differentiating
streptococcal or staphylococcal pharyngitis).
 If the swab cannot be plated immediately it
should be kept moistened with sterile serum –
bacilli remain viable.
 Serum slope shows growth in 4-8 hrs.
 Tellurite medium incubated for 2 days before
considering negative.
 Colony identified by morphological and
biochemical reactions.
 Demonstration of toxicity / Virulence Test:
 Any isolate of the diphtheria bacilli should be
tested for virulence or toxigenicity for the
bacteriological diagnosis to be complete.
 Virulence testing may be by invivo or invitro
methods, the former by the subcutaneous or
intradermal test and the latter by the
precipitation test or the tissue culture test.
 Invitro Test
 Elek’s gel precipitation test : A rectangular strip
of filter paper impregnated with diphtheria
antitoxin (1000 units/ ml) is placed on the
surface of a 20% normal horse serum agar in a
petri dish while the medium is still fluid. When
the agar is set, the surface is dried and narrow
streaks of the strain are made at right angle to
the filter paper strip. A positive and negative
control should be put up. The plate is incubated
at 37°C for 24 – 48 hours.
 Toxin produced by the bacterial growth will
diffuse in the agar and where it meets the
antitoxin of optimum concentration, will
produce a line of precipitation . The presence
of arrow head lines of precipitates indicates
that the strain is toxigeneic. No precipitate will
form in the case of nontoxigenic strains.
 Tissue culture test : The toxigenicity of
diphtheria bacilli can be demonstrated by
incorporating the strains in the agar overlay of
the cell culture monolayers. The toxin produced
diffuses into the cells below and kills them.
 In vivo tests:
 Subcutaneous- Growth from an overnight
culture on Loeffler’s serum slope is emulsified
in 2-4 ml broth and 0.8 ml is injected
subcutaneously into 2 guinea pigs – one of
which has been protected with 500 units of
diphtheria antitoxin 18-24 hrs previously.
 If strain is virulent – unprotected animal will die
within 4 days.
 Method not used usually- wasteful of animals.
 Intracutaneous test:
 Broth emulsion of overnight culture is
inoculated Intracutaneously into 2 guinea pigs -
each receives 0.1 ml in 2 different sites.
 One animal act as a control- receive 500 units
of antitoxin the previous day.
 The other Is given 50 units of antitoxin
intraperitoneally 4 hrs after skin test- inorder to
prevent death.
 Toxigenicity determined by inflammatory
reaction at the site of injection – progressing to
necrosis in 48-72 hrs in the test animal and no
change in control animal.
 Advantage- no animals die.
 Prophylaxis:
 Diphtheria can be controlled by immunisation.
 3 methods of immunisation are available:
active, passive, combined.
 Only active immunisation can provide herd
immunity. Passive and combined can provide
emergency protection in susceptible persons.
 Active immunisation:
 2 antitoxin preparations are used now- Formol
toxoid and adsorbed toxoid.
 Formol toxoid- prepared by incubating the toxin
with formalin at pH 7.4-7.6 for 3- 4 weeks at
37⁰C.
 Adsorbed toxoid – purified toxoid adsorbed to
insoluble aluminium compounds – much more
immunogenic – intramuscular injections.
 Diptheria toxiod is usually given in children as a
trivalent preparation containing tetanus toxoid
and pertussis vaccine also (DPT – triple vaccine)
 In toxoid preparations the lower dose is indicated
by small letter ‘d’ and full dose by capital ‘D’. The
tetanus diphtheria vaccine for adults containing
lower dose diphtheria toxoid is referred to as Td
vaccine.
 Schedule- 3 doses of DPT given at intervals of at
least 4 weeks or 6 weeks , followed by a 4th dose
about a year afterwards. A further booster dose is
given at school interval.
 Passive immunisation- subcutaneous injection of
500-1000 units of antitoxin.
 Combined- first dose of adsorbed toxoid on one
arm, while ADS is given on other arm.
 Followed by full course of active
immunisation.
 Treatment consists of antitoxic and antibiotic
therapy.
 Antitoxin should be given immediately when
diphtheria is suspected.
 The bacterium is sensitive to penicillins.
 Erythromycin is more active in treatment of
carriers.