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…….