1. Corynebacterium

2. Corynebacterium • Classification • Corynebacterium  diphtheriae  and diphtheroids (look  like  C. diphtheriae)  are  Gram- positive, club  shaped  rods.   • Some  are saprophytic • Some   produce  disease   in   animals.   • C. diphtheriae is the most important pathogen in the group. • Morphology and cultural characteristics • Small G+B; arrangement=palisade or Chinese • letters • Growth on B.A – raised, translucent, gray colonies 

3. Diphtheroids Gram stain

4. Arrangement of C. diphtheria

5. Corynebacterium • Loeffler‘s agar slant­ contains serum and egg that enhance the  formation  of metachromatic  granules (polymerizedpolyphosphoric acid) in C. diphtheriae. • Also called Babes-Ernst granules. • They  are visualized  by  staining  with methylene blue.

6. Methylene blue stain from Loefflers slant

7. Corynebacterium • A medium containing tellurite should be used to select for Corynebacterium  and  other G+  organisms -it  inhibits  G­ organisms. Two kinds are used: • Cystine tellurite ­ has alonger  shelf  life • Tinsdale ­ helps  to  differentiate amongst the Corynebacterium. • Colonies on  either  appear black  or gray due to tellurite reduction. • S. aureus  and Listeria  also grow as black colonies.   • On  Tinsdale  C. diphtheriae,  ulcerans, and pseudotuberculosis  form  brown halos around the colonies due to formation of ferric sulfide.

8. Corynebacterium • 3 morphological  types of C. diphtheriae are  found on tellurite containing media: • Mitis – black colonies with a gray  periphery • Gravis – large,  gray  colonies • Intermedius – small, dull gray  to black. • All produce an immunologically identical toxin. • Incubation -35-370 C for 24 hours. • They prefer a pH of 7.8-8.0 for good growth. • They require access to oxygen (poor AnO2 growth). • Biochemistry • Catalase +

9. Corynebacterium • Nonmotile • C. ulcerans is urease +, C. diphtheriae is -, and C. pseudotuberculosis is usually + • Virulence factors­ C. diphtheriae • For C. diphtherias to cause diphtheria an exotoxin must be  produced. • Is a heat-labile polypeptide produced during lysogeny of  a  phage that carries the "tox” gene. • Alkaline pH of  7.8- 8.0, aerobic conditions, and a low environmental iron level are essential for toxin production (occurs late in the growth of the organism). • The  toxin  inhibits protein  synthesis  by  ADP-ribosylating elongation factor  2. • What other organism produces a similar toxin? 

10. Corynebacterium • Trypsin  cleaves  the toxin into 2 fragments, A and B, that are linked together by a disulfide bridge. • Fragment B is required for toxin  binding to tissue cells and fragment A contains  the toxic activity. • One molecule of toxin can inhibit  90%  of the  protein synthesis in a cell. • Systemic effects  include heart failure, paralysis and adrenal hypofunction leading to an Addison’s like disease. • C. ulcerans and C. pseudotuberculosis sometimes make a diphtheria-like  toxin.

11. C. diphtheria toxin • Toxin enters through receptor mediated endocytosis • Acidification of endocytic vesicle allows A to dissociate from B • A enters cycoplasm

12. C. diphtheria toxin

13. Corynebacterium • To prove  that an isolate can cause diphtheria, one  must demonstrate  toxin  production. • This is most often done  on  an  Elek plate: • The  organism is streaked on a plate containing low iron. • A filter strip containing anti-toxin antibody is placed perpendicular to the streak of the organism. • Diffusion of the antibody into the medium and secretion of the toxin into the medium occur. • At the zone of equivalence, a precipitate will form.

14. Elek plate

15. Corynebacterium • Guinea pig or tissue culture toxicity assays may also be done. • Capsule – is protein in nature • Cord factor – is a complex glycolipid (trehalose 6,6’-dicorynemycolate) that has been shown to disrupt mouse mitochondria. • It has not been shown to play a role in the production of diphtheria.

16. Corynebacterium • Clinical Significance (C. diphtheria) • Is normally found in the throats  of  healthy carriers. • The organism infects only man and it has a limited capacity to invade. • Diphtheria -  Disease usually starts as a local infection of the  mucous membranes causing a membranous  pharyngitis • Local  toxin  effects result  in degeneration of epithelial cells. • Inflammation, edema, and production of a pseudomembrane composed of fibrin clots, leukocytes,  and dead epithelial cells and microorganisms occurs  in  the throat.

17. Diphtheria - pseudomembrane • This may obstruct the  airway  and result  in suffocation.

18. 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.

19. Corynebacterium • Prevention- Active immunization with toxoid (alum precipitate). • Is part of the DPT vaccine. • Shick skin test­ like the Dick test in that it  tests  for circulating  antibody  to the toxin by injecting a small amount  of  toxin intradermally and observing for  a  local erythematous  and  necrotic reaction. • If this occurs it indicates  that  the person has no anti-toxin  antibodies  and  is, therefore, susceptible to diphtheria. •  Other Corynebacterium­ are part of the normal flora of  the skin and URT.

20. Corynebacterium • Are called diphtheroids and may occasionally cause disease, particularly in immunocompromised individuals. • C.  ulcerans­  toxigenic strains  may  produce  a  disease similar to, but less severe than diphtheria. • J-KGroup­  commonly  cause  infections  in  those   with underlying disease.   • Diseases include bacteremia, meningitis, peritonitis, wound infections, etc.   • It  is becoming more and more of a problem. • C.  pseudotuberculosis ­ found in those with exposure to animals. • Can cause pneumonia or lymphadenitis. • Produces a different exotoxin than C. diphtheriae.