Aerobic Non-Spore Forming Gram-Positive Bacilli

1. Aerobic Non-Spore Forming Gram-Positive Bacilli Corynebacterium

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

3. Species of Corynebacteria Corynebacterium diphtheriae • Other Significant Corynebacterium species • C. xerosis • C. pseudodiphtheriticum • C. pseudotuberculosis • C. jekeium, (skin) • C. ulcerans Normal flora of RT, urethra, vagina, Skin

4. Corynebacterium spp • Gram positive bacilli, with characteristic morphology (club shaped and beaded) • Non motile • Non spore forming • Non capsulated • Non--hemolytic on sheep blood agar • Facultative anaerobic • C. diphtheriae is fastidious while diphtheriods are non-fastidious • Catalase positive • Oxidase negative

5. ØLipid-rich cell wall contains meso-diaminopimelic acid, arabino-galactan polymers, and short-chain mycolic acids • ØLysogenicbacteriophage encodes for potent exotoxin in virulent strains

8. Corynebacterium: Natural Habitats • Many species normal commensals of the human skin (including • C. amycolatum, • C. jeikeium, • C. urealyticum) • C. jeikeium and C. amycolatum also present in the inanimate hospital environment

9. Corynebacterium: Modes of Infection • Corynebacteriumjeikeium, C. amycolatum, and C. urealyticumas skin flora can be introduced systemically from infected catheter wounds • Corynebacteriumurealyticumas an anterior urethral commensal can invade the urinary tract in debilitated patients

10. Corynebacterium: Types of Infectious Disease • C. jeikeium, C. amycolatum, and C. urealyticum produce wound infection, bacteremia, and endocarditis in hospital patients. • C. urealyticum is a urease producer that causes to deposition of ammonium magnesium phosphate crystals and damages bladder mucosa with ulceration and infection.

11. Corynebacterium: Types of Infectious Disease • Toxigenic strains of C. diphtheriaelyso- genized by tox+-prophage most often cause diphtheria. • Occasional strains of C. ulcerans and C. pseudotuberculosis also produce toxin, but only C. ulcerans is associated with diphtheria-like illness. • Tox–strains of C. diphtheriae cause pharyngitis and endocarditis.

12. Cased by C. diphtheriae Mycocarditis, neuroitis, 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

13. Clinical Forms of Diphtheria • Respiratory • Acquired by droplet spray or hand to mouth contact • Non-immunized individuals are susceptible • Non-respiratory • Systemic • Skin and cutaneous forms

14. C. diphtheriae: Causative Agent of Diphtheria • Respiratory disease–diphtheria • Incubation period–2 to 5 days • Symptoms: sore throat, fever, malaise • Toxin is produced locally, usually in the pharynx or tonsils • Toxin causes tissue necrosis, can be absorbed to produce systemic effects • Forms a tough grey to white pseudomembrane which may cause suffocation

15. . diphtheriae pseudomembrane • WBC + organism • This may obstruct the  airway  and result  in death caused by a lack of air or oxygen

16. Corynebacterium • The more dangerous  effects  occur when the toxin becomes systemic and  attacks the  heart(heart failure),  peripheral nerves (paralysis), and the adrenal glands (hypofunction). • Cutaneous  diphtheria­  More  common  in tropical and subtropical areas. • Necrotic lesions with occasional formation of a local pseudomembrane occur. • Antibiotic susceptibility and treatment • Antiserum ­ once the toxin has bound, however, the antiserum  against it is ineffective. • Penicillin­ to eliminate the organism.

17. Virulence Factors in Corynebacterium Species Phospholipase D: increse vascular permeability and promote spread of organism

18. Corynebacterium: Resistance to -Lactam Drugs Corynebacteriumamycolatum, C. jeikeium, and C. urealyticum are characteristically resistant to penicillin and other -lactam drugs, and uniformly vancomycin susceptible

19. Toxin consists of two fragments • A: Active fragment • Inhibits protein synthesis • Leads to cell/tissue death • B: Binding • Binds to specific cell membrane receptors • Mediates entry of fragment A into cytoplasm of host cell • One molecule of toxin can inhibit  90%  of the  protein synthesis in a cell. • Systemic effects  include heart failure, paralysis and adrenal hypofunction • C. ulcerans and C. pseudotuberculosis sometimes make a diphtheria-like  toxin.

20. C. diphtheria toxin • Toxin enters through receptor mediated endocytosis • Acidification of endocytic vesicle allows A to dissociate from B • A enters cyctoplasm and inhibit protein synthesis by rection with EF

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

22. 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 • Deep blue or red metachromatic granules (accumulated inorganic polyphosphates) by methylene blue stain Loefflers serum

23. The colonies of C. diphtheriae are small, granular, grey, smooth, and creamy with irregular edges

24. Cultural characteristics • On blood tellurite agar • It is selective medium for isolation of C. diphtheriae (Potassium tellurite)

26. 3 biotypes of C. diphtheriaeare 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.

27. Morphology • 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) • Gram stain: • C. diphteriae are gram positive bacilli arranged in Chinese letters form often club shaped

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

29. 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

30. Detection of toxin: 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

31. Filter paper saturated with diphtheria antitoxin Lines of precipitations • 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 Inoculated M.O. Positive Elek’s Test

32. Treatment • Infected patients treated with anti-toxin and antibiotics • Anti-toxin produced in horses • Antibiotics have no effect on circulating toxin, but prevent spread of the toxin • Penicillin drug of choice with erythromycin