microbiology corynebacterium-11 fcm (1).pptx

CORYNEBACTERIUM
Presented By :
Rahul
Shreya
Rimsha
Akshita

CONTENTS
INTRODUCTION MORPHOLOGY CULTURAL
CHARACTERISTI
CS
BIOCHEMICAL
REACTIONS
TOXIN RESISTANCE PATHOGENESIS LABORATORY
DIAGNOSIS
PROPHYLAXIS SCHICK TEST TREATMENT

INTRODUCTION
 Gram – positive, non – acid fast, non – motile rods
 Frequently show club – shaped swellings { hence, named “corynebacterium” (coryne means
club)}
 Most important member of the genus is
n “CORYNEBACTERIUM DIPHTHERIAE”,
the causative agent of the diphtheria.

MORPHOLOGY
Are pleomorphic, non-capsulated, thin, slender and gram –
positive bacilli
Measure about 3-6 µm x 0-.6-0.8 µm
 club shaped ( presence of metachromatic granules)
 bacilli are usually seen in angular fashion, resembling letters
V or L ( called as Chinese letter or cuneiform arrangement)
Bacilli look green and metachromatic granules appear bluish
black on Albert staining

CULTURAL CHARACTERISTICS
Grown best on media enriched with “BLOOD, SERUM OR EGG”
Aerobic and facultatively anaerobes
Optimum temperature for growth is 37ºC & optimum pH is 7.2
Media employed for cultivation of diphtheria bacillus are:
HISS’S SERUM WATER
LOEFFLER’S SERUM SLOPE
TELLURITE BLOOD AGAR MEDIUM

HISS’S SERUM WATER
Liquid medium containing serum
Growth is seen as a turbidity
and pellicle formation

LOEFFLER’S SERUM SLOPE
 Diphtheria bacilli grows rapidly on this medium
 Colonies can be seen in 6-8 hours of incubation
colonies are small, circular, white or creamy
and glistening

TELLURITE BLOOD AGAR MEDIUM
Potassium tellurite selective agent
Organisms grow slowly on this medium and
form grey or black colonies
Colonies may take two days to appear on this medium
Based on colony morphology on tellurite medium and other properties, three
main biotypes can be seen- gravis, intermedius and mitis can be distinguished

BIOCHEMICAL REACTIONS
 Ferment glucose and maltose with the production of acid but without gas
 GRAVIS: ferments starch and glycogen
 INTERMIDIUS AND MITIS: have no such action
 Reduces nitrates to nitrites
 Do not hydrolyse urea or form phosphatase
 Gelatin is not liquefied

TOXIN
• Due to production of a powerful exotoxin
• 90-95% gravis and intermedius strains are toxigenic
• Only 80-85% of mitis strains are toxigenic
• Most widely used strain for toxin production -“PARK-WILLIAMS 8 STRAIN”
PATHOGENECITY
• Depends on presence of a tox gene
• Non-toxigenic strains may be rendered toxigenic, by the process
oflysogenicorphage conversion
• TOXIGENECITY REMAINS AS LONG AS THE BACTERIA IS LYSOGENIC
• When bacteria is freed of it’s phage, by growing it in the presence of
antihpage serum, IT LOSES THE TOXIGENICITY & BECOMES NON-
TOXIGENIC STRAIN.
TOXIGENECITY

RESISTANCE
Readily dies when exposed to a temperature of 58ºC for 10 minutes or 100ºC for
1 minute
Resistant to drying
Susceptible to penicillin, erythromycin and broad spectrum antibiotics

PATHOGENESIS
 Diphtheria is mostly seen in 2 to 10 years of children
 Infection is confined to humans only
 Incubation period is 3 to 4 days
 Infection occurs by way of droplet spread
 Diphtheria is of the following clinical types :
 Faucial
 Laryngeal
 Nasal
 Conjunctival
 Otitic
 Vulvovaginal
 Cutaneous (mainly around mouth and nose)
FAUCIAL
 Most common
• toxin has both local as well as systemic effects
LOCAL
• Bacilli remain confined to the site of entry
• Toxin causes local necrotic changes along with
superficial inflammatory reaction
• Mechanical complications are due to the
pseudomembrane
SYSTEMIC
• Diphtheria toxin releases into blood and
causes to xaemia
• Toxin acts systemically on the cells of several
tissues

LABORATORY DIAGNOSIS
• Laboratory confirmation is necessary for control measures and epidemiological studies but not for the
treatment of cases
• Laboratory diagnosis consists of isolation of organism and demonstration of it’s toxicity
ISOLATION
Culture
Colony
morphology &
Albert’s staining
Biochemical
reactions
Collection of
specimen
Direct
Microscopy
Loeffler’s
serum slope
Tellurite blood
agar
Blood agar
VIRULENCE
TESTS
In vivo tests
Subcutaneous
test
Intracutaneous
test
In vitro tests
Elek’sgel
precipitation test

ISOLATION OF ORGANISMS
 CO LLECTIO N OF SPECIMEN
 two swabs from the le sions are collected
1. sm ear exam inati on
2. c ulture
 swabs are collecte d prior to start of antibiotic s and application of
anti septics
 Swabs are rubbed over the affected area and pseudomembrane, if
form ed
 If there’s no definite localized lesion, swabs should be rubbed
over tonsils and poste rior phar yngeal wall

 Direct Microscopy
 smears are stained with both Gram and Albert
stain
 Albert staining: diphtheria bacilli shows
beaded slender
green rods in typical Chinese letter pattern
 Gram staining: done to identify Vincent’s
spirochaetes and fusiform bacilli

 CULTURE
 Swabs are inoculated in the following culture media:
a) Loeffler’s serum slope
o Growth appears within 6-8 hrs
o Subculture from Loeffler’s is made on tellurite blood agar
o Plate is incubated at 37ºC for 48 hours
b) Tellurite blood agar
o Incubation at 37ºC for at least 48 hours before declaring these as negative, as
growth can sometimes be delayed
c) Blood agar
o useful for differentiating streptococcal or staphylococcal pharyngitis, which can
stimulate diphtheria

COLONY MORPHOLOGY AND STAINING
 Loeffler ’s seru m slope:
o Colon ies are small, circu lar, wh ite or creamy
 Tellu rite b lood agar:
o Diphth eria b acilli grow as b lack or grey colou red colon ies
 Albert stainin g :
o green b acilli with bluish b lack metach romatic gran u les is
seen
 Gram stain ing :
o reveals g ram positive b acilli

Biochemical reactions
 Hiss’s serum water is used for testing fermentation of carbohydrates
*only gravis biotype is positive
GLUCOSE
A
LACTOSE
-
MANNITOL
-
SUCROSE
-
MALTOSE
+
NO3 REDUCTION
+
INDOLE
-
UREASE
-
PHOSPHATASE
+
CATALASE
+
OXIDASE
-
GLYCOGEN*
+
STARCH*
+

20
VIRULENCE TESTS
 Demonstrate the production of exotoxin by
bacteria isolated on culture
 Can be done by two methods:
1) IN VIVO TESTS
2) IN VITRO TESTS

VIRULENCE TESTS
In vivo tests
oSubcutaneous and intracutaneous test
Guinea pigs and rabbits
 In vitro tests
Elek’s gel precipitation test and tissue culture tests

SCHICK TEST
 An intradermal test
 Done to demonstrate circulating diphtheria antitoxin
 Antitoxin may be present either due to previous infection or immunisation
 Results are read after 1, 4 and 7 days
 There could be four types of reaction
o Positive reaction
o Negative reaction
o Pseudoreaction
o Combined reaction

o Positive reaction
 signifies that the person is susceptible to diphtheria due to either absence or lack of
adequate amount of circulating antitoxin
o Negative reaction
 Indicates that toxin has been neutralised by sufficient amount of antitoxin present in the
blood and that the person is immune to diphtheria
o Pseudoreaction
 Indicates that the individual is immune to diphtheria and also hypersensitive to the
components of diphtheria bacilli
o Combined reaction
 Indicates that the individual is susceptible to diphtheria & hypersensitive to bacillus
 Necessary to immunise such persons but vaccine may likely to induce reaction
*SCHICK TEST IS NOT USED NOWADAYS

PROPHYLAXIS
1. Active Immunisation
o Started at 6 weeks of by toxoid in combination with tetanus toxoid and pertussis vaccine
(DPT, Triple vaccine)
o 3 doses are given by intramuscular route at an interval of 4-6 weeks
o Booster doses of DPT are given at 18 months and at 5 years
2. Passive Immunisation
o 500-1000 units of antitoxin [ anti diphtheric serum, ADS ] is administered subcutaneously
3. Combined immunisation
o All persons receiving ADS as prophylactic measure should receive this type of
immunisation

TREATMENT
o Erythromycin (orally or by injection) for 14 days (40 mg/kg per day
with a maximum of 2 g/d) or,
o Procaine penicillin G given IM for 14 days ( 300,000 U/d for patients
weighing < 10 kg and 600,000 U/d for those weighing > 10 kg)
o Patients with allergies to penicillin G or erythromycin can use
rifampin or clindamycin
t

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