Clinical Microbiology=MLS-CMIC-226 Corynebacterium ...In-vivo toxigenicity test: a. Animal...
Transcript of Clinical Microbiology=MLS-CMIC-226 Corynebacterium ...In-vivo toxigenicity test: a. Animal...
Clinical Microbiology=MLS-CMIC-226
Corynebacterium
Diphtheriae and non
Diphtherial Corynebacteriabatch 9
U. Fatima ElShaikh ……0916583988
By the end of this lecture you will
be able to:
Drow a classification scheme of Gram positive bacilli.
Determine the Medical Importance species of
Corynebacterium.
to identify the main characters of Corynebacterium
species.
To discusse the Pathogenicity of Corynebacterium
species.
To determine all the identification test used in the lab. To
diagnose Corynebacterium infections
Classification:G +ve Bacilli
Aerobic and Facultative
anaerobes Strict anaerobic
Non spore forming Spore forming
Clostridium speciesActinomycetes
species
Non Branching Branching
Actinomycetes species
Nocardia species
Streptomycetes species
Spore forming
Non spore forming
Bacillus species
Catalase -ve
Catalase +ve
Erysipellotherix species
Lactobacillus species
Corynebacterium species
Listeria monocytogenes
Mycobacterium species
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Corynobacterium:-
Medical Importance:-
- Non-pathogenic (Commensals)
diphtheroids bacilli – Skin
– URT
– External ear
– Conjunctiva
- Pathogenic:- Human – diphtheriae bacilli
Animals – other species
Corynebacterium species
General properties:
They are Pleomorpic G+ve bacilli found bind to each
other like Chinese litter??????.
Non motile, Non spore forming Non capsulated, aerobic or
facultative anaerobes.
They form Polyphosphate (volutin or metachromatic)
granules when cultured on highly enriched media.
They are highly resistant to drying.
Species of medical importance:
C. diphtheriae. Cause diphtheria
Diphthroides. Opportunistic pathogens that
cause infection to immunocompromized
individual.
C. ulcerance and C. pseudotuberculosis Cause
Diphtheria like illness (usually in a milder form).
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C. diphtheriae: biotypes
biotypes: define biotyping????
Gravis: sever
Intermedius: intermedium
Mitis: mild
*note: their names are given according to the severity of the symptoms.
they can be differentiated at the laboratory by biochemical tests.
What is diphtheria?
Pathogenicity
Pathogenicity
Diphtheria is an infection of pharynex thatcharacterized by formation of gray whitepseudomembrane which consist of inflammatorycells, dead tissue and bacilli which may blockthe respiratory tract leading to Asphexia.
This disease occurred due to Exotoxins whichproduced from the bacilli after infection withprophage β which contain tox gene.
This toxins spread through the blood causedestruction of cardiac, kidney and nervoustissue by inhibition of Elongation factor 2leading to inhibition of protein synthesis.
The toxin have 2 fragments A & B. Fragment Benhance entrance of fragment A to the cell,and Fragment A inhibit protein synthesis.
A-subunit target: EF2, which is a catalyst for hydrolysis of
GTP, required for movement of ribosomes on mRNA.
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C. diphtheriae: Clinical aspects
- Incubation period is usually 2-5 days, during which time the organism becomes established on the epithelial cells of the URT.
- The organism have two distinct components:
(1) a local infection characterized by edema, and the formation of pseudo membrane composed of fibrin, leukocyte exudates, white blood cells, and bacteria.
(2) a profound toxemia which affects the heart and peripheral nerves.
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Pseudomembrane:
- The pseudo-membrane is often thick and very adherent and can cause respiratory obstruction in infants & young children's.
- Removal of the pseudo-membrane increase the absorption of toxin and should not be done prior to the administration of anti-toxin unless necessary to maintain airway.
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C. diphtheriae:
Occasionally the primary infection at skinwound (atypical case) which will lead to mildsystemic infection.
Non-toxigenic (non-pathogenic) of all varietiescan occur.
C. diphtheriae is not naturally pathogenic toanimals, but guinea-pigs and rabbits arehighly susceptible to toxigenic strains or theirtoxins.
Primary infection at skin wound (atypical case) - Diphtheria
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C. diphtheriae: Carriers
Carrier state
- Carriers play an important role in spread of the disease.
- Throat and Nose are the common sites of carriage. Sometimes ear and others occur.
Lab. diagnosis
Specimens:
Throat swab, nasopharyngeal swab or skin swab
Direct examination:
Gram stain showing G+ve bacilli arrange in Chinese
letter.
Inoculation of the specimens on Loffler's serum media or
Dorset egg media for 6 hours and stain fixed smear by
Albert stain or Neisser’s stain or toluidine blue. The
volutine granules stained dark green to black in Albert
stain, dark blue to black in Neisser’s stain and red-
purple in toluidine blue stain.
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Metachromatic granulesAlbert's stain
Neisser’s stain
Culture:
Blood agar: Produce small gray or white convex colonies, non
hemolytic.
Selective media is Tellurite Blood agar TBA (contain 0.03%-0.04%
K. tellurite) and Modified Tinsdale’s medium MTM (contain K.
tellurite + cystein).
Loeffler serum, Dorset egg medium Not for primary isolation.
Incubation:
At 370C in aerobic condition, (20-400C)
Colonial morphology:
Tellurite blood agar: Corynebacteria can
take up the tellurite in the medium and
reduce it to tellurate, which precipitates out
resulting in the observed black colonies. C.
garvis and mitis produe β haemolysis.
Modified tinsdale’s media produce brown
hallow due to H2S production.
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Corynebacterium diphtheriae
Colony morphology on Tellurite BA agar - three cultural variants (biotypes) These can be distinguished on the basis of their colony morphology on tellurite BA agar. The biotypes are gravis, mitis, and intermedius.
Gravis colonies are large, dull, flat, and gray to black.
Mitis colonies are smaller, glossy, and more domed shaped.
Intermedius colonies are very small and may or may not be glossy.
The manifestations of diphtheria bare no constant relationship to the colonial types.
Biochemical characters:
Catalase +ve.
Oxidase and Urease –ve.
Ferment glucose and maltose with acid
production.
C. gravis ferment starch and Terihalose.
C. ulcerance are urease +ve and liquefy
gelatin.
Name this test??
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C. diphtheriae:
Biochemical reaction
StarchSucroseMaltoseGlucose
+-++Gravis
--++Intermedius
--++Mitis
+-++C. ulcerans
IN A DIFFERENT WAY
Specimens: throat and nasopharyngeal swabs
containing part of pseudomembranous.
Direct gram stain: are gram positive rod (Chinese
letters).
Culture: C. diphtheriae are an aerobes and facultative
anaerobes. Temperature range for growth is 20-40ºC
with an optimum of 35-37ºC.
Blood agar: colonies are small, non haemolytic.
Tellurite Blood agar: selective medium for
C.diphtheriae, colonies are black.
Loeffler serum medium: is an enrichment medium,
enhance production of voltine granules.
C. Diphtheriae on Cysteine tellurite agar
Gram stain from plate: gram positive rod, markedly
pleomorphic, long, thin, and curved forms.
Alberts's stain: is used to demonstrate the
metachromatic.
Catalase test: positive produce
catalase enzyme.
Oxidase test: negative.
Rapid Carbohydrates
Utilization Test: Ferment
glucose and maltose with acid
production. A few strains of
gravis and mitis biovars ferment
sucrose.
Glc Mal Lac Suc
•Elek gel precipitation test: for toxogenecity is used
to determine whether the organism is able to produce
the diphtheria toxin or not.
•Or can detect by the PCR amplification of tox gene
Antitoxin is used to neutralize C. diphtheriae toxin.
C. diphtheriae is sensitive to the majority of antibiotics, suchas the:
Penicillin’s
Ampicillin
Cephalosporins
Quinolones
Chloramphenicol
Tetracycline’s
Cefuroxime
Trimethoprim.
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C. diphtheriae: Toxins
Toxin production:
- depend on lysogenic relationship between C.
diphtheria and bacteriophage.
- Strain that lack the phage are non-toxigenic.
antitoxin
A subgroup of antisera usually prepared from the serum of horses immunized against a particular toxin-producing organism.
such as botulism antitoxin and diphtheria antitoxin given prophylactically to prevent those infections.
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To manufacture diphtheria antitoxin, ...
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Toxigenicity test
1. Invitrotoxigenicity test:
(ELEK’S gel precipitation reaction):
Toxigenic strain will secrete toxins that react with antitoxin in the filter paper producing precipitin line.
We need:
Elek’s plate media.
Tested organism
Control strain (Known toxoginic strain).
Sterile filter paper impregnated in antitoxin.
Procedure?????
Results (interpretation of results)?????
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ELEK’S DOUBLE DIFFUSION
2. In-vivo toxigenicity test:
a. Animal toxigenicity test:
Inoculation of toxigenic strain to guinea pig will lead
to death of animal after 48 hours.
Procedure:
2 guinea pig; immunize one with antitoxin
Inoculate both with test organism
After 2 days?????
b. Schick test
Intradermal test used to detect immunization to
diphtheria.
0.2 ml of Highly diluted diphtheria toxin is injected
intradermally in the arm, and heat inactivated toxin is
injected on the other hand.
Interpretation:
• swelling and redness >10cm………+ve test
• swelling and redness 10-5cm………repeat the test
• swelling and redness <5 cm………-ve test
SCHICK TEST49
prevention
Immunization (DPT) Triple vaccine
Please refer to Clinical Microbiology Manual
Assignment
Thank you…….