1. Corynebacterium

2. Corynebacterium Classification Morphology and cultural characteristics
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 (polymerized polyphosphoric 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 a longer  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 Biochemistry
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 C for 24 hours.
They prefer a pH of for good growth.
They require access to oxygen (poor AnO2 growth).
Biochemistry
Catalase +  

9. Corynebacterium Virulence factors­ C. diphtheriae 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   , 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 Capsule – is protein in nature
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 Antibiotic susceptibility and treatment
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-K Group­  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.