Corynebacterium

Diphtheria

1. CORYNEBACTERI
UM
DIPHTHERIAE
Dr V S Vatkar
Asso Prof
Microbiology Department
D Y Patil Medical College, Kolhapur

2. CORYNEBACTERIUM



Kingdom:Bacteria
Phylum:Actinobacteria
Order:Actinomycetales
Family:Corynebacteriaceae
Genus:Corynebacterium
Species:C. diphtheriae

3. HISTORY:
BRETONNEAU(1826):
RECOGNIZED AS CLINICAL ENTITY, WHO CALLED IT AS
DIPHTHERITE.

Greek word koryne means club & Diphtheros meaning
leather i e leathery pseudo memb over tonsils.
Diphthria bacillus first observed & described by
Klebs(1883) & First cultivated by Loeffler(1884): Hence known as
Klebs-Loeffler bacillus.
Roux & Yersin (1888): show pathogenic effect of diphtheria toxin
Its antitoxin : described by Von Behring in 1890

4. CORYNEBACTERIUM
DIPHTHERIAE
Morphology:
Slender rod with tendency of clubbing at one or both
ends
Measuring 3-6µm ×0.6-0.8µm.
Pleomorphic
Nonsporing, nonmotile, noncapsulated.
Cells often show septa, and branching is infrequently
observed.

5. Bacilli being at various angles to each other, resembling
the letter V or L Called Chinese letter or
cuneiform arrangement due to bacilli divides
horizontally & daughter cell lie at acute angle to parent
cell (snapping type of division)
Granules: composed of polymetaphosphotase seen in
the cells & present at the ends or poles of the bacilli,
metachromatic granules also called volutin or Babes
Ernst granules.(polar bodies)
Granules are more strongly Gram positive than rest of
the cell.
Loeffler’s methylene blue: granules take bluish
purple colour
MORPHOLOGY

6. Albert’s, Neisser’s and Ponder’s
special stains for demonstrating granules
clearly.
Well developed in enriched media like
blood agar or Loeffler’s serum slope

7. GRAM STAINING
ARRANGEMENT OF
BACILLI

8. STAINING WITH METHYLNE BLUE :

9. ALBERT STAIN

10. •CULTURAL CHARACTERISTICS :
Media:
growth scanty on ordinary media.
Enrichment with blood, serum or egg is necessary for good growth.
Loeffler’s serum slope & tellurite blood agar used for cultivation.
Loeffler’s serum slope Tellurite blood agar
colonies within 6-8 hrs -small, colonies takes 2 days to appear
circular white opaque discs, enlarge gray or black colonies
on incubation acquire yellow tint reduce tellurite to metallic
tellurium
Optimum temperature:
for growth is 37° (15-40°C)
Optimum pH: 7.2
Aerobe and facultative anaerobe
Modifications such as McLeod’s and Hoyle’s media have been utilised

11. •A medium containing tellurite should be used to select for
Corynebacterium and other Gm+ organisms. It inhibits
Gm­ organisms.
•Two kinds are used:
•Cystine tellurite ­ has a longer shelf life
•Tinsdale ­ helps to differentiate amongst the
Corynebacterium.
Colonies on either appear black or gray due to
tellurite reduction.
S. aureus and Listeria also grow as black colonies.

12. Black colonies on potassium tellurite medium

13. LOEFFLER’S SERUM SLOPE

15. Based on colony morphology on tellurite
medium & other properties, McLeod classified
diphtheria bacilli into three types:
Gravis: causing most serious disease
Mitis: the mildest variety
Intermedius: disease of intermediate severity.

16. Gravis and intermedius types are associated with high
case fatality rates.
Paralytic complications common with gravis,
hemorragic complication are common with gravis and
intermedius.
Obstructive lesions in air passage in mitis infections.
Diphtheria bacilli ferment glucose, galactose, maltose
and dextrin producing acid but no gas
Some strains ferment sucrose.
Hiss serum water used for testing sugar fermentation.

17. Gravis Gravis Intermedius Intermedius Mitis Mitis
MorphologyMorphology Short Short
rodsrods,uniform ,uniform
staining,pleomo-staining,pleomo-
rphism.rphism.
Long barred Long barred
forms,clubbed forms,clubbed
ends,very ends,very
pleomorphic.pleomorphic.
Long,curved,Long,curved,pleple
omorphic omorphic
rods,prominent rods,prominent
granules.granules.
Colony on Colony on
tellurite agartellurite agar
In 2-3 days,flat In 2-3 days,flat
colonies with colonies with
raised dark raised dark
centre centre and and
crenated edge crenated edge
with radial with radial
striation- ‘striation- ‘daisy daisy
headhead’’ colony colony
Dull granular Dull granular
centre with centre with
smoother, more smoother, more
glistening glistening
peripheryperiphery and and
lighter ring near lighter ring near
the edge- the edge- ‘‘frog’sfrog’s
egg’egg’ colony colony
In 2-3 In 2-3
days,colonies days,colonies
becomebecome flat, flat,with with
a a central central
elevation elevation
‘‘poached egg’ poached egg’
colonycolony
Consistency of Consistency of
coloniescolonies
Like ‘Like ‘cold cold
margarine’,margarine’,
brittle, move as a brittle, move as a
whole on whole on
plate,not easily plate,not easily
picked out or picked out or
emulsifiableemulsifiable
Intermediate Intermediate
between gravis between gravis
and mitisand mitis
Soft, buttery and Soft, buttery and
easily easily
emulsifiableemulsifiable

18. TABLE CONTINUED ………..
HomolysiHomolysi
ss
variablevariable Non hemolyticNon hemolytic Usually Usually
hemolytichemolytic
Growth in Growth in
brothbroth
Surface pellicleSurface pellicle, ,
granular granular
deposit, little or deposit, little or
no turbidityno turbidity
Turbidity in 24Turbidity in 24
hourshours, clearing , clearing
in 48 hours, with in 48 hours, with
fine granular fine granular
sedimentsediment
Diffuse turbidityDiffuse turbidity
with soft pellicle with soft pellicle
later later
Glycogen Glycogen
and and
starch starch
fermentatifermentati
onon
Positive Positive Negative Negative Negative Negative

19. DIPHTHERIA TOXIN:
Virulent strains of diphtheria produce a very
powerful exotoxin.
Pathogenic effects of the bacillus are due to
the toxin.
95-99 % of gravis and intermedius are
toxigenic.
80-85 % of the mitis strains are toxigenic.
The strain universally used for toxin
production is the ‘park williams 8’ strain
This strain described as mitis by Topley and
Wilson
and as intermedius strain by Cruickshank

20. TOXIN (MW 62000)
Fragment AFragment A Fragment B Fragment B

Molecular weightMolecular weight2400024000 38000 38000
Function Function Enzymatic Enzymatic
activityactivity
Binding toxin to the cellsBinding toxin to the cells
It is a protein.
Both fragments necessary for toxic effect. When released from
bacterium,toxin is inactive because active site on fragment A is
masked.
Probably Proteases present in culture medium or infected tissues
activates the toxin.
Toxin is labile. Prolonged storage, incubation at 37°C for 4-6
weeks,
treatment with 0.2- 0.4 percent formalin or acidic pH converts it
to toxoid.

21. •TOXIN PRODUCTION DEPENDS
UPON:
Corynephages (tox +):
acts as genetic determinant controlling toxin production.
Non toxigenic strains may be rendered toxigenic by
infecting them with beta phage or some other toxlarger
phage- known as lysogenic conversion.
when bacillus cured of its phage,it loses toxigenic capacity.
 Concentration of iron:
optimum level of iron for toxin production is 0.1 mg per
litre.
Conc. Of 0.5 mg per litre inhibits formation of toxin

22. ACTION: Toxin acts by ihibiting
protein synthesis.
Fragment A inhibits
polypeptide chain
elongation in presence
of nicotinamide
adenine dinucleotide
(NAD) by inactivating
elongation factor 2
(EF2)

24. RESISTANCE :
Colonies remain viable for two or more weeks
at 25-30°C.
Readily destroyed by heat in 10 minutes at
58°C. and in a minute at 100°C.
Resistant to action of light, dessication and
freezing.
It has been cultured from dried bits of
pseudomembrane after 14 weeks.
Remains virulent in blankets, floor dust for 5
weeks.
Easily destroyed by antiseptics.
Susceptible to penicillin, erythromycin and
broad spectrum antibiotics.

25. ANTIGENIC STRUCTURE :
Antigenically heterogenous.
Gravis strains classified into 13 types.
Intermedius into 4 types.
And mitis into 40 types.
Gravis strains of:
 Types I and III common in Great Britain,
Type II worldwide,
Type IV mainly in Egypt,
Type V in USA.

26. TYPING
1.Bacteriophage typing: There are 15 bacteriophage types have
been described.
 Types I and III are mitis,
 IV and VI are intermedius,
 VII is avirulent gravis,
 Remainder are virulent gravis.
2.bacteriocin(diphthericin) typing:
3.Bacterial polypeptide analysis:
4.DNA restriction patterns and hybridisation
with DNA probes

27. PATHOGENESIS:
Incubation period 3-4
days.
 Carriers have longer
incubation period
Route of entry: upper
respiratory tract.
Adhere by pillus
shaft(spa A) and two
small pili(spaB & spaC)
Grow locally & produce
toxin

28. SITE OF INFECTION:
1.Faucial: commonest type
2.Laryngeal
3.Nasal
4.Otitic
5.Conjunctival
6.Genital- vulval, vaginal or prepucial.
7.cutaneous

29. BULL NECK
PSEUDOMEMBRANE

30. CLINICAL TYPES :
Malignant or hypertoxic:
Severe toxemia with marked adenitis (bull neck)
Death is due to circulatory failure.
Septic:
leads to ulceration,cellulitis and even gangrene
around pseudomembrane.
Hemorrhagic:
Bleeding from the edge of the membrane,
epistaxis, conjunctival hemorrhage, purpura and
generalised bleeding tendency.

31. COMPLICATIONS:
Asphyxia due to mechanical obstruction
Acute circulatory failure
Post diphtheritic paralysis:
seen in 3
rd
or 4
th
week of disease
palatine or ciliary but not pupillary
Septic : pneumonia and otitis media
Relapse in 1% cases.

32. LABORATORY DIAGNOSIS:
1.Isolation of diphtheria bacillus:
One or two swabs from lesion collected under vision
Gram staining, Leishman staining, done.
Inoculation done on Loeffler’s serum slope, tellurite
blood agar and on ordinary blood agar.
Colony morphology seen after incubation.
Colony smear done & methylene blue staining,
neisser or Albert staining done to see metachromatic
granules.

33. 2.VIRULENCE TESTS :
In vivo tests:
1.Subcutaneous test:
0.8 ml of emulsified growth in
broth injected s.c. into two
guinea pigs. One of which is
protected with 500 units of
antitoxin 18-24 hrs
previously. Both animals
observed.
2. Intracutaneous test :
0 .1 ml of emlsified growth in
broth in injected
intracutaneously in two
guinea pigs. One given 500
units and other given 50
units of antitoxin.
In vitro tests:
1.Elek’s gel
precipitation test:
2. Tissue culture test:
incorporating the strains in
agar overlay of cell culture
monolayers. Toxin produced
diffuses into the cells below
and kills them.

34. ELEK’S GEL PRECIPITATION TEST:

35. PROPHYLAXIS:
Immunisation:
1.Active immunisation:
Formol toxoid and adsorbed toxoid.
Available as DPT or along with inactivated polio
vaccine
Active immunisation started at 6 weeks of age by
toxoid in combination with tetanus toxoid and
pertusis vaccine. i.m. route.at 4-6 wk interval.
Booster given at 18 months and at 5 years.

36. PASSIVE IMMUNITY :
Emergency measure when susceptible are
exposed to infection.
Consists of subcutaneous administration of
500-1000 units of antitoxin.(antidiphtheritic
serum, ADS)
This is horse serum, precaution against
hypersensitivity should be observed.
Combined immunisation :
Administration of first dose of adsorbed toxoid
in one arm, while ADS is given in other arm.
Followed by full course of active immunisation.

37. TREATMENT :
Antitoxic and antibiotic therapy.
Antitoxin given immediately in suspected cases.
Recommended dose is 20,000 to 1,00,000 units for
serious cases.
Half dose if given i.v.
C. diphtheriae sensitive to penicillin and can be
cleared from throat within few days of treatment.
Erythromycin effective in carriers.

38. OTHER PATHOGENIC
CORYNEBACTERIA :
C. ulcerance:
can cause diphtheria like lesion.
it liquefies gelatin, ferments trehalos, does not reduce
nitrate to nitrite.
Arcanobacterium hemolyticum cause pharyngitis, skin
ulcers.
C. Jakeium cause cutaneous and blood infections
Corynebacteria causing superficial skin
infections:
C. minutissimum
C.tenuis

39. DIPHTHEROIDS:
Normal commensals in throat, skin,
conjunctiva and other areas.

e.g. c.pseudodiphtheriticum.
C. xerosis.