Corynebacterium diphtheria
- Gram positive, non-acid fast, non-motile rods, irregularly stained
and with granules.
- Club shaped swelling at one end- hence the name
Corynebacterium
- C.diphtheriae- most important species of this group.
- It causes Diphtheria.
- In ancient times it was called Egyptian or Syriac ulcer.
- Diphtherite – tough leathery, tenacious pseudomembrane which
was described by Bretonneau in 1826
- It is called Kleb’s or Loeffler’s bacillus because in 1883, Kleb’s
observed it first and Loffler isolated it in 1884.
- Loeffler also conducted various experiments on animals by
injecting cultivated organisms and concluded that some diffusible
product is responsible for the disease.
- Exotoxin was discovered by Roux and Yersin in 1888 and they
established the pathogenic effect.
- Antitoxin was described by von Behring in 1890.
-
MORPHOLOGYSlender rods, club shaped in one or both ends. 3-6μ x 0.6-0.8μ
They are pleomorphic, non-sporing, non-acid fast, non-capsulated
and non-motile.
Cells have septa and branch sometimes
Gram positive but tend to get decolorized quickly and easily
Contain granules of polymetaphosphate- role uncertain
- If stained by methylene blue, granules take up bluish purple or
reddish purple color. Hence they are called meta-chromatic
granules.
- These are also called Volutin or Babes-Ernst granules –usually
situated in the poles of the bacilli. Hence also called ‘polar bodies’
- Stains used- Methylene blue, Albert’s, Neisser’s, and Ponder’s- to
demonstrate granules easily and clearly.
- Because of incomplete separation during division the organisms
are attached to each other at various angles or ends. Hence they
appear as alphabets “V” or “L” – called Chinese letter or
cuneiform pattern.
CULTURE- Can grow on ordinary media but scanty. So special media enriched
with blood, egg or serum used.
- Optimum temperature- 37°C (15-40°C), ph7.2
- Aerobe and facultative anaerobic.
- Loeffler’s serum slope, tellurite blood agar and its modifications
and McLeod’s and Hoyle’s medium are used
- Loeffler’s serum slope- grows rapidly 6-18 hours which is before
any of the contaminants.
- Small colonies appear at first, later bigger, opaque, circular and
with yellow tint.
- Tellurite medium- 0.04% tellurite inhibits the growth of other
bacteria, acting as a selective agent. Diphtheria bacilli reduce
tellurite to metallic tellurium giving the colonies a black grey
color.
-
-
McLeod ClassificationBased on the tellurite agar growth- Gravis, intermedius and mitisbiovars.
Nowadays not used since it is not consistent and also it is more
important to know if the strain is more toxigenic or not and typing
of strain is to be done for prevention and epidemiology.
Usually with gravis and intermedius- grave case fatality
Gravis- paralytic complications are more
-
-
Gravis and intermedius – hemorrhagic complications are more.
Mitis – obstruction to air passage
Mitis is usually seen in endemic areas.
Gravis and intermedius in epidemics.
Mitis establishes commensal relationship more easily.
Also gravis and intermedius types usually spread more in
immunized and naturally immune people.
Biochemical propertiesFerment sugars with production of acid only (not gas). Hiss serum
water is used for fermentation reactions.
Glucose, maltose, galactose, dextrin are fermented
Lactose, mannitol and sucrose not fermented.
Urease and phosphatase negative
Toxin –
- Exotoxin produced cause the clinical effects
- Toxigenicity of the strain depends on a bacteriophage called beta
phage or tox+ phage.
- Lysogenic or phase conversion renders the non-toxigenic strain,
toxigenic
- If you grow a toxigenic strain i.e. tox+ strain in anti-phage or
antitoxin serum the strain will lose its Toxigenicity.
- “Park Williams 8” strain is used for the toxin production
everywhere commercially
- Gravis and intermedius isolated in 95-99% of acute cases and
mitis only in about 80-85% are toxigenic.
- All effects of Diphtheria are due to the toxin
- Avirulent strains are usually seen in convalescent carriers and
extra faucial infections
- Toxigenicity is controlled by the phage gene.
- Invasiveness is controlled by the bacterial genes.
- All toxigenic strains of C.diphtheriae give out exotoxin. In vitro
production of toxin depends on
a) iron concentration b) osmotic pressure c) amino acid
concentration 4) pH and 5) availability of suitable carbon and
nitrogen.
- Factors in vivo not fully understood
-
-
Diphtheria toxin –
Heat labile polypeptide with a molecular wt of 62,000. Lethal
dose- 0.1μg/kg
Has 2 fragments- when the disulphide bond is split- fragment A
and fragment B
Fragment B -mol.wt.-38,000, transports fragment A into the cell.
Fragment A –mol.wt.- 24,000 inhibits polypeptide chain
elongation, in presence of NAD (nicotinamide adenine
dinucleotide) by inactivating the elongation factor-2 (EF-2)
EF-2 is required for the translocation of polytpeptidyl- transfer
RNA from acceptor to donor site of eukaryotic ribosome.
Toxin fragment A inactivates EF-2 by catalyzing a reaction that
yields free nicotinamide plus an active adenosine diphosphate
ribose EF-2 complex. This leads to the abrupt stoppage of protein
synthesis leading to the necrotizing and neurotoxic effects of
diphtheria toxin on the cells especially myocardium, adrenals and
nerve endings
- Same type of exotoxin can be produced by some of the
Pseudomonas aeruginosa strains.
- Toxin can be toxoided for immunization.
-
-
Resistanceviability 2-3 weeks at 25-30°C
destroyed by heat at 58°C for 10mins, 100°C for 1 min
antiseptics easily destroy it
resistance to light, desiccation, freezing than other bacilli
Virulence not lost in fomites like blankets, floor dust, toys, pencils
etc.
Susceptible to penicillin, erythromycin, broad spectrum antibiotics
Aerosol spread leads to infection to others
Antigenic structureHeterogenous
Gravis- 13 types, intermediate- 4 types, mitis- 40 types by
agglutination
Different countries have different types.
Bacteriophage types- 15 types
Type I, III – mitis
Type IV , VI – intermedius
Type VII – gravis avirulent
Rest – gravis virulent
Other methods of typing-
-
Diphthericin typing- bacteriocin typing(colicin)
Bacterial polypeptide analysis
DNA restriction patterns
Hybridization by DNA probes.
Pathogenicity –
Incubation period- 3-4 days.
Can be as short as 1 day.
In carriers- prolonged
Site- faucial, laryngeal, nasal, otitic, conjunctival, genitovulval, vaginal,
prepucial, cutaneous- usually secondary on pre-existing skin lesion
Diphtheric Whitlow or ulcer may occur on skin. Usually non-toxigenic
variety cause
Faucial- commonest type- mild catarrhal inflammation to varied
symptoms
Clinically1. Malignant or hypertrophic: toxemia, adenitis (bull neck). Death
due to circulatory failure (paralytic sequelae in recovered
patients)
2. Septic: ulceration, cellulitis and sometimes gangrene around the
pseudomembrane.
3. Hemorrhagic: bleeding from edge of membrane, epistaxis,
conjunctival hemorrhage, purpura and generalized bleeding tendency
Complications1. Asphyxia- tracheostomy maybe required due to the obstruction by
pseudo membrane
2. acute circulatory failure  peripheral, cardiac
3. Post-diphtheric paralysis- third or fourth week of disease.
Palatine and ciliary paralysis can occur ,which usually recovers.
4. Septic  pneumonia, otitis media. 1% cases relapse.
Exotoxin- causes local necrosis (division of bacteria and release of
exotoxin)  fibrinous exudates.
- Exudate mixes with epithelial cells, leucocytes, RBCs and bacteria
forming pseudo membrane
- Immunized individual also can have infectious by non-toxigenic
diphtheria since immunity is to toxoid and not bacilli. This is
usually mild.
Guinea pig- subcutaneous injection of toxigenic strain- death of animal
in 2-4 days
1. Gelatinous, hemorrhagic edema, necrosis at the site of inoculation
2. Swollen, congested draining lymph nodes
3. Peritoneal exudate
4. Congested, abdominal viscera
5. Enlarged and hemorrhagic adrenals
6. Pleural exudate
7. Pericardial effusion
Lab diagnosis-
- Only for epidemiological purpose.
- Immediate treatment to be started- antitoxin and antibiotics
1. Direct examination of swab from pseudomembrane
- Stain the smears- look for others which mimic e.g. Vincent’s
spirochete- Gram’s, Leishman stain
- Albert’s, Neisser’s, Ponder’s or Methylene blue
- Immunofluorescence
- Preliminary report to be given
2. Plate another swab in Loeffler’s serum slope- 4-16 hours
Observe the colonies  stain by Methylene blue, Albert’s, Ponder’s,
Neisser’s
3. Potassium tellurite medium – to be kept minimum of 2 days.
More commonly used for screening convalescents, contacts and
carriers since it inhibits the growth of other bacteria in the specimen
inoculated.
4. Blood agar- differentiates Streptococcal pharyngitis
Once confirmed strain should be tested for virulence.
In vivo methods- not useful
Subcutaneous test-: 2 guinea pigs
inject growth emulsified in
broth
Protected with antitoxin
18-24hrs previously
unprotected
die within 2-4days
Autopsy (character-
mention)
Intracutaneous (intradermally)
- Guinea pig or rabbit – inoculate 2 animals
Control
Test
Receives 500 units of
Of antitoxin 24hrs before test
Result- no change
50 units of antitoxin
after test
result- necrosis at site of
Injection in 48-72hrs
- No death of animals in both cases
- 10 strains can be tested at a time on a single rabbit
In vitro test- Elek’s Gel Precipitation Test.
1.
Rectangular piece of filter paper
dipped in 1000 units of antitoxin
placed before agar sets
Media in 20% normal horse serum
agar.
2.
Non toxigenic strain (neg- control)
Test strain
Toxigenic strain (positive- control)
3.
Non-toxigenic
Test strain (toxigenic)
(Arrow-head precipitation lines)
Positive control
2. Tissue Culture tests- Agar + cell mono layer below
- Strain of diphtheria inoculated into the agar
- Toxin diffuses to the cells and destroys them.
- Shows that the strain is toxigenic
Epidemiology- Pediatric disease
- Developed countries freed and rare instances of the disease seen.
- Due to the well organized immunization program.
- Epidemic has been seen during 1990’s in the erstwhile Soviet Union.
Endemicity –
- Age –less commonly seen in first year due to the passive immunization
from mother
- 2-5 yrs age group affected most
- 5-10 yrs- less due to the active immunity by subclinical infection
-Asymptomatic carriers are important source of infection- nasal and throat
carriage
- Skin infections are also a source, but they tend to provide natural immunity
- Natural disease seen mostly in humans, rarely in cows.
Prophylaxis- Active immunity is important to improve the overall immunity in a
community (Herd immunity).-DPT,DT etc- Expanded program of
Immunization.
- Passive and Combined immunity provide emergency protection
Susceptibility test- Schick test is useful in developed countries where infection is
rare.
Active – Von Behring 1913 (risky)
Danyz phenomenon- toxin- antitoxin combines in varying proportion to retain
potency. If they are in equal amounts, the combination is not potent.
Treatment –
- Antitoxin (ADS) 20,000-1,00,000 units (1/2 IU)
- Antibiotic to reduce carriage and convalescent relapses (hypersensitivity
reaction)
Homework- standardization of toxins and antitoxins
-active immunization (DPT) (DT etc)
(Standardization)
Others –
C.ulcerans - Related to diphtheria can cause Diphtheria like lesions. Resembles Gravis
but liquefies gelatin, ferments trehalose slowly, nitrate negative.
- 2 types of toxin – one resembles Diphtheria toxin
- Other one is C.pseudotuberculosis
- Guinea pig- lesion resembles that of C. diphtheria
- Humans get infection by drinking the milk from infected cows.
- Sensitive to erythromycin
- ADS is protective.
OthersArcanobacterium hemolyticus- pharyngitis, skin ulcers
C.jeikeium- cutaneous and blood infections in immune compromised
Multi drug resistant and susceptible to vancomycin
C. pseudotuberculosis- Preisz- Nocard bacillus
Usually causes lung infections in sheep and
suppurative lymphadenitis in horses.
C.renale- cystitis and pyelonephritis in cattle
C. equi – pneumonia in fowls
Superficial skin infectionErythrasma – localized infection of stratum corneum in axilla and groin
C.minutissimum – lipophilic, grown in media with 20% fetal calf serum
-
Trichomycosis axillaries – pigmented nodules around axillary and pubic hair
– C. tenuis
DIPHTHEROIDSDiphtheroids resemble C.diphtheriae, can be mistaken for it, hence called
Diphtheroids.
These are the normal flora of throat, skin and conjunctiva
Diphtheroids stain more uniformly
Possess few metachromatic granules
- These have a palisade arrangement
- Some are indistinguishable
- Biochemical and virulence tests can be used to differentiate the species
reliably
e.g. C.pseudodiphtheriticum also called C.hofmanii – throat infections,
they do not ferment glucose and are urease positive.
C.xerosis – conjunctival sac infections, urease negative
- Both pyrazinamidase positive (C.diphtheria negative)
Others – Propionibacterium – P.acnes, P.granulosum, and P.avidum
present in skin
Anaerobic, aero tolerant, grow in lipid containing media.
Acne lesion – P.acne has been isolated, but role uncertain.
C.parvum – is a immunomodulator used in research.