Sharq Elneil College

School of Medical Laboratory Sciences

Department of Microbiology

Medical Bacteriology course

U.Mahadi Hassan Mahmoud Bsc, Msc, MIBMS Microbiology

The bacterium that causes diphtheria was first described by Klebs in 1883, and was cultivated by Loeffler in 1884, who applied Koch's postulates and properly identified Coryneb-bacterium diphtheriae as the agent of the disease.

In 1884, Loeffler concluded that C. diphtheriae produced a soluble toxin, and thereby provided the first description of a bacterial exotoxin

G +ve Bacilli

Aerobic and Facultative

anaerobes Strict anaerobic

Non spore forming Spore forming

Clostridium species Actinomycetes

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

Mycobacterium species

Listeria monocytogenes

C. diphtheriae. Cause diphtheria

Diphthroides. Opportunistic pathogens that cause infection to immunocompromized individual.

C. ulcerance. Cause Dimphtheria like illness

C. pseudotuberculosis. Cause Diphtheria like disease.

Species of medical importace:

They are Pleomorpic G+ve bacilli found bind to each other like Chinese litter or Arranged in V forms or Palisades

Non motile, Non spore forming Non capsulated, aerobic or facultative anaerobes.

i. Loeffler's Serum Slope.

ii. Tellurite BA.

- Temp. 37oC,

- Aerobic and Facultative AnO2.

They form Polyphosphate (volutin or metachromatic) granules when cultured on highly enriched media.

They are highly resistant to drying.

They are fastidious organism that need Blood, serum or egg for growth.

solely among humans

spread by droplets

secretions

direct contact

Poor nutrition

Crowded or unsanitary living conditions

Low vaccine coverage among infants and children

Immunity gaps in adults

Diphtheria is an infection of pharynex that characterized by formation of gray white pseudomembrane which consist of inflammatory cells, dead tissue and bacilli which may block the respiratory tract leading to Asphexia.

This disease occurred due to Exotoxins which produced from the bacilli after infection with prophage β which contain tox gene.

This toxins spread through the blood cause destruction of cardiac, kidney and nervous tissue by inhibition of Elongation factor 2 leading to inhibition of protein synthesis.

The toxin have 2 fragments A & B. Fragment B enhance entrance of fragment A to the cell, and Fragment A inhibit protein synthesis.

Largely controlled now by vaccination

However, factors such as poverty and other

social factors have led to diphtheria being an

endemic/epidemic in many regions of the

world

Epidemiology

Specimens: Throat swab.

Direct examination:

Gram stain showing G+ve bacilli arrange in Chinese letter.

Inoculation of the specimens on Lofflere’s serum media or Dorset egg media for 6 hours and stain fixed smear by Albert stain or Neisser stain. The volutin granules staind dark green to black in Albert and dark blue to black in Neisser stain.

Culture:

Blood agar.

Selective media is Tellurite Blood agar (contain 0.03%-0.04% K. tellurite) and Modified Tinsdale’s medium (contain cystein).

Incubation:

At 37 C in aerobic condition.

Colonial morphology:

B.A Produce small gray or gray white convex colonies.

Tellurite blood agar produce black colonies due to formation of K. tellurate, C. garvis, mitis and ulcerance produe β haemolysis.

Modified tinsdale’s media produce brown hallow due to H2S production.

Biochemical characters:

Catalase +ve.

Oxidase and Urease –ve.

Ferment glucose and maltose with acid production.

C. gravis ferment starch and Terialose.

C. ulcerance are urease +ve and liquefy gelatin.

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 with low iron concentration.

Tested organism

Control strain (Known toxoginic strain).

Sterile filter paper impregnated in antitoxin.

Procedure.

Results (interpretation of results).

Invivotoxigenicity test:

Inoculation of toxigenic strain to guinea pig will lead to death of animal after 48 hours.

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:

______________________________________________________________________ Result Test Arm Control Arm Interpretation Diphthri

(Toxin) Inactivated Toxin Immunization 36h 120h 36h 120h ______________________________________________________________________ Negative -- -- -- -- Immune, Not Not Hypersensitive Required

----------------------------------------------------------------------------------------

Positive + + -- -- Not Immune, Not Required Hypersensitive -------------------------------------------------------------------------------------------------------------------

-- Negative + -- + -- Immune Not Hypersensitive Required -------------------------------------------------------------------------------------------------------------------

-- Positive & + + + -- Not Immune Contra- Pseudo Hypersensitive indicated ______________________________________________________________________

Penicillin

Erythromycin

Gentamicin

Sanitary: Reduce carrier rate by use of vaccine.

Immunological: A vaccine (DPT) prepared from an alkaline formaldehyde inactivated toxin (i.e. toxoid) is required. Passive immunization with antitoxin can be used for patients.

For therapy of

DT

tumor

tumors

!!