Corynebacterium

diphtheriae

Anav
 Anav

Coryneform (Gk: koryne- a club)

 club-shaped  Corynebacterium
pleomorphic Gram  Aureobacterium
positive rods  Cellulomonas
 Microbacterium
 Oerskovia
 arranged in 'V' form  Sanguibacter
or palisades  Brevibacterium
 Turicella
 Dermabacter
 Corynebacterium

 Order- Actinomycetales
 Family-Corynebacteriaceae
 Genus- Corynebacterium

 Classification on the basis of pathogenicity:

 1. Pathogenic - Corynebacterium diphtheriae
C. ulcerans
 2. Non-pathogenic - C. pseudodiphtheriticum (nasal mucosa)
C. xerosis (conjunctival sac)
C. pseudogenitalium (genitourinary tract)
Corynebacterium diphtheriae
(diphtheros: leather)
 "Klebs-Loeffler bacillus"

Morphology:
- Thin, slender Gram positive rods that are highly pleomorphic
- 3-5m X 0.5-0.8 m
- One of the shapes they assume is club-shape due to the presence of volutin granules at
one or both ends
- Arranged in angular fashion or palisade form resembling 'Chinese letters'
- Non-sporing, non-capsulate, nonmotile

Staining characters:
- Although Gram positive, they are readily decolorised except for the granules
- Loeffler's alkaline methylene blue- granules are reddish purple; cytoplasm-lightly stained
=>metachromatic granules
- Albert's stain - granules- greenblack and the bacteria will appear green
- Non acid fast
C.diphtheriae
 Cultural characteristics:
 - Aerobic or facultatively anaerobic, optimum temp 370C in media enriched
with blood, serum or egg (pH 7.2)
 - Usual media:
 a) Loeffler's serum slope
 b) Tellurite containing media –
 Hoyle's medium
 Downie's medium
 Modified Tinsdale Medium
C.diphtheriae
Biotypes
- Based on the colonial morphology on the tellurite medium, and other morphology,
McLeod classified diphtheria bacilli into 3 types-gravis, intermedius and mitis.

Characters / Biotypes Gravis Intermedius Mitis

Colony appearance 1-2 mm diameter 0.5-0.7 mm diameter 1.5-2 mm diameter

after 18-24 hrs on Dry, grey, coherent Smooth, grey Grey, smooth and
Tellurite blood agar “Daisy head” colonies “Frog’s egg” colonies glossy surface
“Poached egg”
colonies

Hemolysis Variable Non-hemolytic Usu. Hemolytic

Microscopic morphology -Tear drop form -Canoe or cigar shaped -Pleomorphic with
of on Hoyle’s medium - Some pleomorphic - Highly pleomorphic classic morphology
(methylene blue stain)

Halo formation on Brown halos around Brown halos after 48 Brown halos after 24
Modified Tinsdale individual colonies hours hours
Medium
C.diphtheriae

 Cultural characteristics:
C. diphtheriae cysteinase

Potassium tellurite
(from media)
Cystine (in media) H2S

BROWN
HALO
 C.diphtheriae

RESISTANCE AND ANTIGENIC STRUCTURE

 Resistance  Antigenic
 Readily killed by moist heat in 10 structure
minutes at 60OC.
 Resist natural drying and may
remain viable in dust for weeks if  K Ag- Heat labile protein
protected from sunlight.
 Sensitive to penicillin and
 O Ag- Heat stable
erythromycin polysaccharide
 C.diphtheriae

TOXIN
 The pathogenicity is due to exotoxin
 The gene for toxin production is carried out into the bacteria by a
bacteriophage.
 Nontoxigenic strains may be converted to the toxigenic state by
infection with tox+ corynephage.

 Toxin consists of 2 fragments- “O’Meara’s factors- Fragment A and

B
 Fragment B- Concerned with receptor mediated endocytosis
 Fragment A- Concerned with necrotic lesions

Heparin binding- Epidermal growth factor (HB-EGF)

is the receptor in susceptible host cell

Toxin (DT)
 R domain
 T domain
 C domain
C.diphtheriae
ACTION OF TOXIN
C.diphtheriae
PATHOGENESIS
MODE OF INFECTION- Respiratory (Droplet) / Extrarespiratory (Discharge)
 C.diphtheriae

PATHOGENESIS
 Primarily a childhood disease (2-14 yrs)
 Incubation period 3-4 days
 Diphtheria - faucial, laryngeal, nasal,
conjunctival, vulvovaginal, otitic, cutaneous
around mouth and nose
 Initial lesions usu. occurs on the tonsils and
oropharynx and from this site, they may spread
to the nasopharynx, larynx and trachea
 Then production of exotoxin occurs which
causes the necrosis of cells in the area
 Necrosed epithelium + fibrinous exudate +
leucocytes + RBCs + Bacteria

Pseudomembrane
C.diphtheriae

PATHOGENESIS
 The diphtheria toxin diffuses into the blood stream from the primary
site of infection and causes toxemia.
 The toxin has got affinity for MYOCARDIUM, ADRENAL TISSUE
and NERVE ENDINGS.
 Complications: Local – laryngeal obstruction

Systemic – Diphtheritic myocarditis

Paralysis
Degenerative changes in
liver, kidney
C.diphtheriae

LABORATORY DIAGNOSIS
 Specimen collection
 Faucial or nasal diphtheria- both from nose and throat – swab

 Direct microscopy
 Rarely helpful

 C.diphtheriae doesn’t form much volutin when growing in the

throat
 Isolation and identification
 Rub the swab over the moist surface of the Loeffler serum slope

 Plate the swab on the blood agar and on either Hoyle’s or

Tinsdale tellurite blood agar

 Incubate aerobically at 370C.
 C. diphtheriae

LABORATORY DIAGNOSIS
SWAB

Loeffler Slant Sheep Blood Agar Modified Tinsdale Medium (MTM)

Methylene blue stain MTM Smear

Gram stain of each representative colony

Preliminary notification Coryneform morphology

to
Subculture on Biochemical test / Toxigenicity
the clinician Blood agar/ test (Final identification)
Loeffler slant
 C. diphtheriae
In-vivo

LABORATORY DIAGNOSIS - Detection of Toxigenicity In-vitro

 In-vivo test Subcutaneous test
Intradermal test
Subcutaneous test
Action / Subject Guinea Guinea
pig pig (Test)
(Control)

I/P injection of antitoxin (12 before Given Not given

test)
S/C injection of a suspension in 2- 0.3 ml 0.3 ml
5 ml of nutrient broth of the 18 hr
Loeffler slope culture of the strain
being tested

After 1-4 days Unaffected Die

 C. diphtheriae

LABORATORY DIAGNOSIS - Detection of Toxigenicity

Intradermal test
Action / Subject Guinea pig Guinea pig
(Control) (Test)

Antitoxin (previous day) 500 units -

Intradermal injection of toxin 0.2 ml 0.2 ml

Simultaneous I/P injection of antitoxin - 50 units

After 1-4 days Unaffected Local erythematous

lesion at the site of
injection
C. diphtheriae

LABORATORY DIAGNOSIS - Detection of Toxigenicity- In vitro test

 In-vitro test (ELEK’S AGAR GEL PRECIPITATION TEST)
C. diphtheriae
TEST FOR IMMUNITY – SCHICK TEST
 Intradermal test to demonstrate immunity, i.e.,
circulating diphtheria antitoxin (due to previous
immunization or infection)

 A test dose of 0.2 ml diphtheria toxin containing 1/50

MLD is injected intradermally in one forearm and an
identical amount of inactivated toxin (heated at 700C-30
min) in another.
 C. diphtheriae SCHICK TEST-
Result
REACTION SCHICK SCHICK TEST REMARKS
CONTROL DOZE
DOZE
NEGATIVE No erythema No erythema Immune to diphtheria

POSITIVE No erythema Erythema in 24-48 hrs Susceptible to

& may persist for 4-5 diphtheria
days (1-5 cm in
diameter)
PSEUDO Erythema in Erythema in 6-24 hrs - Both AT & allergy
NEGATIVE 6-24 hrs - subsides in 72 hrs present
(SCHICK subsides in
NEGATIVE) 72 hrs
COMBINED Erythema in Erythema in 6-24 hrs - Susceptible to
REACTION 6-24 hrs - persists for 4-5 days diphtheria &
(PSEUDO+ subsides in hypersensitive to the
POSITIVE) 72 hrs antitoxin
C. diphtheriae

C. diphtheriae
 O
O
C. diphtheriae
Difference between Diphtheria bacillus & Diphtheroids
Criteria / Subjects Diphtheria bacillus Diphtheroids

Metachromatic granules Present Absent

Parallel arrangement Absent Present

Size and shape Long and slender Short and thick

Culture on basic media No growth Growth
MacLeod’s blood Usu. Rough colonies Smooth colonies
Tellurite agar

Sucrose fermentn - +/-

Toxigenicity + -
REVISION
Toxemia/ Bacteremia
Most pathogenic biotype
REVISION
Mechanism of brown halo
REVISION
Albert’s stain result
Why chinese letter
REVISION

Difference between fragment A and B

CAMP TEST (HW)
REVISION

 Media for Corynebacterium

 Difference between diptheroids n c. diphtheriae
REVISION

Babes-ERNST granules N ITS

FUNCTION
REVERSE CAMP TEST (HW)
REVISION

ZN stain-
Gram stain-
REVISION

 PReDilection of ToXiN
REVISION

O’Meara’s Factors
SCHICK TEST
REVISION

Pseudomembrane
Inhibition of CAMP TEST (HW)