This presentation mainly talks about the bacterium Coryebacterium diptheriae the causative agent of Diptheria

1. Study of important properties, pathogenicity and laboratory
identification of Corynebacterium diphtheriae
By
KEVEN LIAM WILLIAM
209701
2ND M.Sc Zoology
ST. ALBERT’S COLLEGE (AUTONOMOUS), ERNAKULAM

2. INTRODUCTION
• Gram +ve, non – acid fast, non – motile rods with irregularly stained segments,
due to the presence of granules in some species.
• Frequently show club – shaped swellings – hence the name Corynebacterium.
• Corynebacterium diphtheriae, the causative agent of diphtheria is the major
pathogen in this group.
• C .ulcerans : diptheria – like lesions.
• C.minutissimum and C.tenuis : causes superficial skin infections.
• Diptheroids : normal commensals in throat, skin and conjunctiva

3. HISTORY
• Hippocrates – provided the first clinical description of diphtheria in the 4th
century B.C.
• Bretonneu (1821), a French army surgeon – described unique clinical
characteristics of the disease, and used the term ‘dipht’ ‘erie’ to signify the
tough leathery pseudo membrane that occurs in oropharynx and sometimes in
nasopharynx.
• Bacterium that caused diphtheria was first described by Klebs in 1883 and was
cultivated by Loeffler in 1884, who applied Koch’s postulates and properly
identified Corynebacterium diphtheriae as the agent of the disease.
• Roux and Yersin (1888) – discovered the diphtheria exotoxin and establised its
pathogenic effects.
• The antitoxin was described by von Behring (1890)

4. Corynebacterium diphtheriae morphology
• Slender, gram – positive rods, pleomorphic; easily decolourised.
• 0.6 – 0.8 μm in diameter and 3.6 μm in length
• Irregular swelling at one or both ends (club – shaped).
• Non – sporing, non – capsulated and non – motile.
• Possess polymetaphosphate granules which serve as storage granules and are called volutin or
Babes – Ernst granules or metachromatic granules.
• Take up bluish purple colour against lightly stained cuytoplasm when stained with Loeffler’s
methylene blue.
• They are often situated at poles – polar bodies.
• Special stains for demonstrating the granules – Albert’s stain, Neisser’s stain, Ponder’s stain.
• Bacilli are arranged in pairs, palisades or small groups
• Bacilli lie at various angles to each other resembling the letters V or L – “Chinese letter
pattern” or “cuneiform pattern”.

5. CULTURAL CHARACTERISTICS
• Aerobe and facultative anaerobe.
• Optimum temperature = 37°C.
• Growth scanty on ordinary media.
• Enrichment with blood, serum or egg is necessary for good growth
• Potassium tellurite (0.04%) acts as a selective agent as it inhibits growth of most oral
commensals and retards the growth of C.albicans and S.aureus.
MEDIA FOR CULTIVATION
• Blood agar
• Loeffler’s serum slope
• Tellurite blood agar
• Hoyle’s tellurite lysed – blood agar
• Tinsdale’s medium (cystine added to tellurite containing agar)

6. COLONY CHARACTERISTICS
• Blood agar – small, granular and gray with irregular edges; hemolysis may or may not be present.
• Loeffler’s serum slope
– Very rapid growth
– Colonies in 6 – 8 hours.
– Initially circular white opaque colonies and acquire yellowish tint on incubation.

7. • Tellurite blood agar:
– Growth slow; colonies seen after 48 hours;
– The colonies are brown to black with a
brown – black halo because the tellurite is
reduced to metallic tellurium;
– Staphylococcus also produce such
colonies.
• Tinsdale medium (also contain cystine in
addition to tellurite)
– Grey black colonies with dark brown
haloes include C.diphtheriae and
C.ulcerans (these contain cystinase)

8. Biotypes of Corynebacterium Diphtheriae (McLeod and Anderson)
Properties Gravis Intermedius Mitis
Morphology
– Short Rods
– Uniform Staining
– Few or no granules
– May be pleomorphic
– Long-barred forms, clubbed
end
– Poor Granulation
– Very pleomorphic
– Long Rods
– curved shaped
– pleomorphic
– Prominent granules
Colony on Tellurite Blood Agar
≥ 2mm, dull greyish black,
opaque colonies, daisy head,
brittle, like cold margarine.
< 0.5 mm, gray colony, dark
center shining surface, frog’s
egg colonies
≥ 2mm, gray, opaque glossy,
smooth surface, poached egg
colonies, soft buttery, easily
emulsifiable
Hemolysis Variable Non-hemolytic Usually Hemolytic
Glycogen and Starch
fermentation
Positive Negative Negative
Antigen Types 13 4 40
Fatality Rate High High Low
Complications Paralytic Hemorrhagic Obstructive
Endemicity Epidemic Epidemic Endemic

9. • gravis and mitis – associated with high case fatality rates
• Paralytic complications more with gravis
• Hemorrhagic complications – gravis and intermedius
• Obstruction to air passage – mitis
• Mitis – endemic
• gravis and intermedius – epidemic
BIOCHEMICAL REACTION
• Hiss serum sugars – for testing fermentation reactions
• Ferments glucose, galactose, maltose and dextrose
• Does not ferment lactose, sucrose and mannitol.
• Proteolytic activity is absent
• Urease test = negative
• Phosphatase test = negative
• Produce cystinase (halo on Tinsdale’s medium)

10. RESISTANCE
• Cultures remain viable for 2 – 3 weeks at 25 – 30°C.
• Destroyed by heat.
• Resistant to light, dessication or freezing
• Easily destroyed by antiseptics.
• Susceptible to pencillin, erythromycin and broad spectrum antibiotics.
ANTIGENIC STRUCTURE AND TYPING
• Serotyping : antigenically heterogenous
– Gravis – 13 types
– Intermedius – 4 types
– Mitis – 40 types
• Bacteriophage typing – 15 types
• Bacteriocin typing – diptheriocin typing

11. VIRULENCE FACTORS
• Virulent strains of diphtheria produce a very powerful exotoxin.
• The ‘virulence’ of diphtheria bacilli is due to their capacity to:
– Establish infection and growing rapidly.
– Quickly establish an exotoxin
• Avirulent strains are common among convalescents, contacts and carriers, particularly those with
extra – faucial infection.
DIPHTHERIA TOXIN
• The pathognomonic effects are due to the toxin.
• Almost all the gravis and intermedius strains and 80 – 85% of mitis strains are toxigenic.
• Toxin is a protein.
• Molecular weight – 62,000
• Two fragments – A and B of MW 24,000 and 38,000 respectively (both are necessary for the toxic
effect)
• Extremely potent – 0.1 μg to guinea pig
• Inactive when released because the active site on fragment A is masked.

12. • Activation is probably accomplished by proteases present in the culture medium
and infected tissues.
• All the enzymatic activity of the toxin is present in the fragment A.
• Fragment B is responsible for binding the toxin to the cells.
• The antibody to fragment B is protective by preventing the binding of the toxin to
the cells.
• The toxin is labile.
• Prolonged storage, incubation at 37°C for 4 – 6 weeks, treatment with 0.2 – 0.4%
formalin or acid pH converts it to toxoid.
• The diphtheria toxin acts by inhibiting protein syntesis.
• Specifically fragment A inhibits polypeptide chain elongation in the presence of
nicotinamide adenine dinucleotide by inactivating EF – 2.

13. Mechanism of action of diphtheria toxin

14. PATHOGENICITY
• Incubation period in diphtheria = 3 – 4 days.
• In carriers, incubation period may be very prolonged.
• The site of infection may be faucial, laryngeal, nasal, otitic, conjunctival, genital – vulval, vaginal or
prepucial and cutaneous.
• Faucial diphtheria is the commonest type.
• According to the clinical severity, diphtheria can be classified into:
– Malignant or hypertoxic – severe toxemia with marked adentitis (bullneck). Death is due to
circulatory failure.
– Septic, which leads to ulceration, cellulitis and even gangrene around the pseudomembrane.
– Hemorrhagic, characterised by bleeding from the edge of the membrane, epistaxis,
conjunctival haemorrhage, purpura and generalised bleeding tendency.

15. • Common complications include:
– Asphyxia due to mechanical obstruction of the respiratory passage by the pseudomembrane
for which an emergency tracheostomy may become necessary.
– Acute circulatory failure, which may be peripheral or cardiac.
– Post diphtheritic paralysis, which typically occurs in the 3rd or 4th week of the disease.
– Septic such as pneumonia and otitis media.
– Relapse may occur in about 1% of cases.
• After infection, the bacilli multiply on the mucous membrane or skin abrasion.
• The toxigenic strains start producing toxin.
• Diphtheria is a toxemia.
• The bacteria confine to the site of entry but the exotoxin is absorbed into the mucous membrane
and causes destruction of epithelium and a superficial inflammation response.
• The toxin causes local necrotic changes.
• The resulting fibrinous exudate, together with the epithelial cells, leucocytes, erythrocytes and
bacteria constitute pseudomembrane.
• Any effort to remove it will tear off capillaries beneath it and causes bleeding.

16. LABORATORY DIAGNOSIS
• This is to confirm the clinical impression and for epidemiological purpose;
• Specific treatment must never be delayed for laboratory reports, if the clinical picture is strongly
suggestive of diphtheria.
• Any delay may be fatal.
• It consists of the isolation of the organism and demonstration of its toxicity.
• Specimens – swabs from nose, throat or other suspected lesions
• Smear examination : Gram stain
– Shows beaded rods in typical arrangement
– Difficult to determine from some commensal Corynebacteria normally found in the throat.
– Albert’s stain or Neisser’s stain is useful for demonstrating the granules.
• If the swabs cannot be inoculated promptly, they should be kept moistened with serum;
• Inoculate on
– Loeffler’s serum slope
– Tellurite blood agar or Tinsdale medium
– Blood agar (for differentiating Staphlyococal or Streptococcal pharyngitis that stimulate diphtheira)

17. • Processing
– Serum slope may show growth in 4 to 8 hours, but if negative may need to be incubated
for 24 hours.
– Smear may show “diphtheria like” organisms.
– By about 48 hours, Tellurite plates will yield growth;
– The isolate must be submitted for – ‘virulence tests’ or ‘toxigenicity tests’ before the
bacteriological diagnosis is complete.
VIRULENCE TESTS
• In vivo methods
– Subcutaneous test
– Intracutaneous test
• In vitro methods
– Elek’s gel precipitation test
– Tissue culture test

18. Emulsify the growth form an overnight culture of
Loeffler’s serum slope in 2 – 4 ml broth
Protected with
500 IU of antitoxin
18 to 24 hours
previously
Unprotected
Test animal
Control animal
0.8 ml injected subcutaneously into two
guinea pigs
Die in 4 days if the strain is virulent,
autopsy shows characteristic features.
Remains healthy
SUBCUTANEOUS
TEST

19. 0.1 ml of emulsion broth inoculated intracutaneously in
two guinea pigs
Control
animal
Test animal
Give 50 IU of antitoxin
intraperitoneally 4 hours after
skin test (to prevent death)
Inflammatory reaction. Progress to
necrosis in 48 to 72 hours
NO CHANGE
INTRACUTANEOUS
TEST

20. ELEK’S GEL PRECIPITATION TEST
• In vitro test
• A rectangular strip of filter paper is saturated with the diphtheria antitoxin (1000
units/ml);
• This strip is placed on agar plate with 20% house serum, while the medium is setting;
• The cultures to be tested are streaked at right angles to the filter strip;
• A +ve and –ve control should be put;
• The plate is incubated at 37°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.
• The presence of such arrowhead lines of precipitates indicates that the strain is
toxigenic.
• No precipitate will form in the case of non-toxigenic strains.

22. TISSUE CULTURE TEST
• Bacteria incorporated into an agar overlay of cell culture monolayers;
• The toxin, if produced, diffuses into the cells below and kills them;
• PCR: Polymerase chain reaction for detection of Toxin gene (tox) has been
developed to detect the presence of genes coding for the toxin, in clinical
isolates.
• ELISA: The test may be done to detect toxin from the patient's isolate using
antitoxin and enzyme-substrate system.

23. EPIDEMIOLOGY
• In endemic areas, it is mainly a disease of childhood.
• It is rare in the first year of life due to the passive immunity obtained from the mother,
• reaches a peak between 2 and 5 years ,
• falls slowly between 5 and 10 years, and rapidly between 10 and 15 years with only very low
incidence afterwards because of active immunity acquired by repeated subclinical infections.
• Asymptomatic carriers who outnumber cases by a hundredfold or more in endemic areas are
the most important sources of infection.
• In the temperate regions, carriage is mainly in the nose and throat.
• Nasal carriers harbour the bacilli for longer periods than pharyngeal carriers.
• In nature, diphtheria is virtually confined to human beings, though cows may on occasion be
found to have diphtheritic infection of the udder.

24. PROPHYLAXIS
• Protective immunity
– Dependent on the levels of antitoxin antibodies present in the circulation.
– Objective of immunisation – increase protective levels of antitoxins in circulation
– Tests done : In vivo Schick (neutralisation – based skin test), In vivo assays (determined
by passive hemagglutination or by neutralisation in cell culture). Antitoxin levels more
than 0.1 IU/per ml of blood is considered an index of protective antibody level.
• Vaccines
– Three methods – active, passive and combined.
– Active immunisation can provide herd immunity and lead to eradication of the disease.
– Passive and combined immunisation can provide emergency protection to susceptible
individuals exposed to risk.
Passive immunisation
• Used when susceptible persons are exposed to infections.
• Consists of the subcutaneous administration of 500 – 1000 units of antitoxin ADS. Since it is a
horse serum, precaution against hypersensitivity should be observed.

25. Active immunisation
• Done using a killed vaccine.
• Two preparations:
– Formol toxoid (fluid toxoid) – prepared by incubating the toxin with formalin.
– Adsorbed toxoid – purified toxoid adsorbed onto insoluble aluminium phosphate and more
immunogenic than formol toxoid.
– Dosage – usually given in children as a trivalent preparation containing tetanus toxoid and
pertussis vaccine such as DTP, DPT or triple vaccine. For young children, it is given in a
dose of 10 – 25 Lf units.
– Schedule - consists of three doses of DPT given at intervals of at least four weeks,
preferably six weeks more, followed by a fourth dose about a year afterwards. A further
booster dose is given at school entry.
– Side effects – injection site reactions (redness, warmth, swelling, tenderness, itching, pain,
hives and rash), fever, drowsiness, fretfulness, vomiting, anorexia, rarely convulsions.

26. Combined immunization
• Consists of administration of the first dose of adsorbed toxoid on one arm, while ADS is
given on the other arm to be continued for the full course of active immunization.
• Ideally, all cases that receive ADS prophylactically should receive combined immunisation.

27. TREATMENT
• Consists of antitoxic and antibiotic therapy.
• Antitoxin – given immediately when suspected – fatality rate increases with
delay in starting antitoxic treatment.
• Recommended dose – 20,000 to 1,00,000 units for serious cases – half the
dose given intravenously.
• Antitoxin treatment – not given in cutaneous diphtheria – causative strains are
non – toxigenic.
• C.diphtheriae are sensitive to penicillin and can be cleared from the throat
within a few days of penicillin treatment.
• Erythromycin is more active than penicillin in the treatment of carriers.
• Diphtheria patients are given a course of penicillin though it only supplements
and does not replace antitoxin therapy.