Aerobic Non-Spore Forming Gram-Positive Bacilli Corynebacterium

Gram Positive Bacilli Gram positive rods Spore forming Non spore forming Anaerobic Aerobic Clostridium spp Bacillus spp Corynebacterium

Corynebacterium spp Gram positive bacilli, with characteristic morphology (club shaped and beaded) Non motile Non spore forming Non capsulated Facultative anaerobic Breakdown glucose by oxidative and fermentative i.e. O+/F+ C. diphtheriae is fastidious while diphtheriods are non-fastidious Catalase positive Oxidase negative

Species of Corynebacteria It caused diphtheria C. diphtheriae is the only pathogenic members of this genus Normal flora of RT, urethra, vagina, Skin

Diphtheria Cased by C. diphtheriae Mycocarditis, neuroitis, palate perforation Acute, Toxin mediated Recovery or complication & death (if more toxin absorbed) Childhood disease affect upper respiratory tract Diphtheria Respiratory obstruction due to extensive membrane formation Transmitted by droplet infection 2-6 days I.P. Sore throat, Pharyngitis 2-3 days, Bluish white adherent pseudo membrane

Specific treatment must be never delayed for laboratory results To confirm the clinical manifestation

Symptomatic patient Asymptomatic

Laboratory diagnosis of case Specimen: A throat swap Culture: The swap is inoculated on Loeffler's serum medium and/or on blood tellurite agar aerobically at 37C for 24. On Loeffler's serum medium: Corynebacteria grow much more readily than other respiratory pathogens Used to enhance the characteristic microscopical appearance of corynebacteria The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges Loefflers serum

Cultural characteristics On blood tellurite agar (Mcloed’s blood agar) It is selective medium for isolation of C. diphtheriae (Potassium tellurite) 3 biotypes of C. diphtheriae are characterized on BTA i.e. Gravis, mitis and intermedius biotypes The most severe disease is associated with the gravis biotype Colony of gravis biotype is large, non-hemolytic & grey. Colonies of mitis biotype are small, hemolytic and black Colonies of intemedius biotype are intermediate in size, non-hemolytic with black center & grey margin.

Morphology Stain Gram +ve, nonspore forming nonmotile bacilli Club-shaped (Coryne= club) arranged at acute angles or parallel to each other (Chinese letters appearance) Beaded (metachromatic granules) Stain Gram stain: C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped Polychrome methylene blue stain: C. diphteriae appears beaded due to the presence of intercellular “Metachromatic or volutin" granules By stain, the granules appear red while the rest of organism appears blue.

Gram stain of C. diphtheriae C. diphtheriae on BTA

Biochemical Reaction All Corynebacterium species are catalase positive (Also, Staphylococcus and Bacillus species are catalase positive)

2- Carbohydrate Fermentation Test: Principle: Each species of corynebacteria has its specific carbohydrate fermentation pattern C.diphtheriae can be differentiated from other Corynebacterium species by fermentation of glucose and maltose but not sucrose with production of acid only Procedure: Inoculate three tubes of carbohydrate fermentation medium (broth containing one type of sugar and phenol red as the pH indicator) with the test organism Glucose Maltose Sucrose Incubate the tubes at 37oC for 24 hrs.

Results: C. diphtheriae C. xerosis Sugar fermentation can be indicated by change of color of the medium from red to yellow due to formation of acid which decrease the pH Glucose Maltose Sucrose Glucose Maltose Sucrose +ve +ve +ve +ve +ve - ve C. diphtheriae C. xerosis

Swap from throat & nose Inoculation on Loeffler’s Or BTA for 24 h/37 C Diphtheria like M.O.

One is used as Test The second is used as Control If both GP live Two guinea pigs are used One is used as Test The second is used as Control Injected with diphtheria antitoxin Both test and control injected with isolated MO If both GP live If control live & test die Diphthrioids 0r non-toxigenic C. diphtheriae i.e. non pathogenic C. diphtheriae i.e. produce exotoxin

In Vitro: Elek’s Test Principle: It is toxin/antitoxin reaction Toxin production by C.diphtheriae can be demonstrated by a precipitation between exotoxin and diphtheria antitoxin Procedure: A strip of filter paper impregnated with diphtheria antitoxin is placed on the surface of serum agar The organism is streaked at right angels to the filter paper Incubate the plate at 37C for 24 hrs

Filter paper saturated with diphtheria antitoxin Resuls: After 48 hrs incubation, the antitoxin diffusing from filter paper strip and the toxigenic strains produce exotoxin, which diffuses and resulted in lines four precipitation lines radiating from intersection of the strip and the growth of organism Lines of precipitations Inoculated M.O. Positive Elek’s Test