Determinative Bacteriology Flashcard

Corynebacterium

Gram positive, straight or slightly curved, thin rods that are slightly tapered or have “club ends”.

Various arrangements, “Chinese letter”  1883 Klebs described the strain  1884 Loeffler proved it caused diphtheria                         Genus contains:

Gram positive, straight or slightly curved thin rods that have club ends, pleomorphic
Aerobic, facultatively anaerobic strains
Most are catalase +, non motile, non sporeforming, and non acid fast.
Most ferment glucose and other carbohydrates.
Peptidoglycan contains meso-diaminopimelic acid
Cell walls contain: mycolic acid, arabinose and galactose.

 

Corynebacterium classification based on?

 

Classification based on the following cell wall structures:
Chemotype IV
Mycolic acids
Straight chain saturated, unsaturated fatty acids
MenaquinonesVit K2

 

Cell Wall Corynebacterium

 

Chemotype IV cell walls- contain meso diaminopimelic acid (meso DAP), arabinose and galactose
Mycolic acids “corynomocolic acids, C-22-36 atoms long
Straight chain saturated and unsaturated fatty acids
Dihydrogenated menaquinones (vit K) with 8 or 9 isoprene units.

 

Habitat Corynebacterium

 

Widely distributed in nature.  Found in soil and water, on humans resides on the skin and mucous membranes
In humans found in nasopharynx,vagina and on skin

 

Corynebacterium diphtheriae

Causative agent of Diphtheria, “skin membrane”.

The microbe produces a potent exotoxin.
A pseudomembrane forms in the airway.
Humans only natural reservoir
2-5 day incubation period
Upper respiratory tract
Number of cases declining since WW II, in 1920 200 cases per 100,000 people,  in 1994, 2 cases reported in the U.S., one of the cases was fatal and in Miami.
Today no longer a serious health problem in developed countries because infants are vaccinated against the microbe.  However in other countries it still remains a problem.

 

Pathogenesis of C. diphtheriae

Disease is spread via a respiratory route, during infection, the strain grows in the nasophayrnx, as the level of Exotoxin increases, causes necrosis of the mucosa, “diphtheritic pseudomembrane” forms.  This is an exudate of WBC’s, RBC’s and dead bacteria in a fibrin network.  This membrane may eventually cover the tonsils, pharynx, larynx, and posterior nasal passages.  The main action site for the toxin is the heart and peripheral nervous system.

The organism may also cause a primary cutaneous infection, in which the toxin is absorbed systemically.   The lesions look like other skin lesions, can be ulcerative “ecthyma diphtheriticum”.
The toxin has a direct effect on the heart, central and peripheral nerve system, liver and kidneys.  Cardiac issues may appear in 20-70% of patients.  Neurologic issues appear in 20-75% of patients.  

 

Isolation and Identification
Corynebacterium diphtheriae

Gram stain

Inoculate the following media:
Blood agar plate
Loeffler serum medium
Tinsdale medium, Potassium Tellurite medium

4 distinct colony types (biotypes) designated: gravis, mitis, intermedius, belfanti, are observed on isolation.

 

4 biotypes of C. diphtheriae

 

Gravis and mitis:  fairly large, convex colonies with entire edges.  Weakly hemolytic on sheep’s blood agar, slightly.
Intermedius:  small, blacker, dense colonies on tellurite containing agar.
All 4 biotypes grow as black colonies with brown halos on modified Tinsdale medium, and all 4 form gray-white, smooth, nonhemolytic colonies on SBA.  

 

Isolation media C.diphtheriae

 

Loeffler’s medium:
Beef Blood serum and dextrose.  This medium enhances storage granule formation, which are polymerized polyphosphate (Babes-Ernst bodies), aka metachromatic granules.  Cells removed from this medium and stained with methylene blue, cells are blue, granules are red.
•Tinsdale’s or Potassium Tellurite medium 
-Potassium tellurite, inhibits the growth of most normal flora in the nasophayrnx.  The corynebactium, reduce the tellurite to tellurium, colonies appear grey black, with a brown halo on this medium.

 

Identification of
Corynebacterium diphtheriae

 

Tests for toxigenicity
Schick test ( in vivo test )
Elek test ( in vitro test )

 

In vivo test C. diphtheriae
Schick test

 

Used during outbreaks of diphtheria to determine which cases are susceptible to the disease.
A small amount of diphtheria is injected id into one forearm and heated toxin into the other.  Susceptibility vs immunity depends on the presence of circulating antibody. 
Redness and induration  1-2 days at test site only or at both sites with absence at control site in 4-7 days signifies susceptibility due to lack of antibody to toxin.
No reaction at both the test and control sites or redness and induration at 1 day and rapid fading from both, signifies immunity.  

 

In vitro test C. diphtheriae

 

Modified ELEK test for toxigenicity
Add medium to petri dish:  rabbit serum, K tellurite, and virulence agar.
Place filter paper with diphtheria antitoxin across center of medium above before it hardens
Inoculate plate 2 hrs post harden, use a positive and negative control, and unknown
24-48 hrs check for white ppt lines extending from Tox+ strains intersect antitoxin, thereby I.D’s + tox producing strains

 

Diphtheria Toxin

 

Produced by C. diphtheriae strains infected by the bacteriophage “Beta phage”.
Toxin, is a 58,342 dalton protein (535 amino acid residues).
Splits into 2 Fragments:  Fragment A and Fragment B. Inactivation of protein synthesis. 
Regulation of the Tox gene is controlled by the interaction of iron in the medium,  as the iron concentration increases, the iron repressor complex inhibits transcription of the Tox gene
Treatment:  DPT, and antibiotics                                     Fragment A, 21,500 daltons

  1          A      193              B      539                     •Fragment B, 37,200 daltons

NH——————————————-COOH
aa—————-aa————————-aa                   

 

Diphtheria toxin

R domain

 

A domain of the B chain, called the R domain, binds a protein receptor on the host cell surface.  This receptor has been identified as heparin binding epidermal growth factor precursor (HB-EGF).  After the toxin has bound the cell receptor, the host cell takes up the toxin in an endocytic vesicle.  In this vesicle the pH drops to 5,  allows for the unfolding of both the A and B chains. This allows regions of both A and B to insert into the vesicle membrane, the disulfide bond between the chains is broken and releases A into the cytoplasm.  The A chain catalyzes the ADP-ribosylation of EF-2, which renders it useless for protein synthesis.  

 

Diphtheria toxin A chain

 

In the cytoplasm the A chain catalyzes the ADP-ribosylation of elongation factor 2 (EF-2).  This (EF-2), is an essential factor of eukaryotic protein synthesis.  Attachment of the ADP-ribosyl group to EF-2 renders it inactive. As a result ribosomes can not form polypeptides, protein synthesis halts, the cell dies.
An extremely potent toxin.  A single molecule of A chain can kill a eukaryotic cell.

 

Diphtheria toxin Structural gene

 

The structural gene encoding this toxin is carried

on a group of lysogenic bacteriophages, (β). 

Production of this toxin is enhanced when the bacteria are grown in low iron medium.
These phages carry the tox genes.
Since these phages are lysogenic the tox genes get incorporated into the chromosome.  
Only tox+ strains of C. diphtheriae produce the toxin.  

 

Regulation of tox

 

The genes for toxin production  (tox) are regulated by the diphtheria toxin repressor (DtxR), this is an iron-activated regulatory element.
Fe+2 does not by itself control the expression of tox, but a corynebacterial protein is involved.  Instead the iron activates the dtxR on the chromosome and represses expression of toxUnder iron limiting conditions, the 2Fe-DtxR-tox operator dissociates and diphtheria toxin is expressed

 

DPT (Prevention of diphtheria)

 

(diphtheria toxoid, pertussis acellular vaccine and tetnus toxoid), 3 intramuscular doses given at 4-8 week intervals to all children beginning at 6-8 weeks of age, with a fourth dose given 6 to 12 months later. 
Travelers to diphtheria-endemic areas should receive booster inoculations. 
Because of a failure of most adults to receive booster inoculations a susceptible population exists, 10-60% of adults lack adequate levels of circulating antibodies.   

 

Treatment of diphtheria

 

Administer antitoxin immediately.  The antitoxin is generated in horses, therefore check for hypersensitivity, it will only neutralize toxin that has not bound target cells.
Administer penicillin or erythromycin to kill growing C. diphtheriae and thus preventing further toxin development. 
Antibiotics prevent a carrier state from developing and secondary infection.

 

Diphtheria as a Disease of Adults

 

Historical:  Colonial America, this disease caused a high adult mortality rate.  George Washington is thought to have died from diphtheria at age 67.  Remember if members of a population had no exposure as a child, they acquired no protective immunity.  It was a new disease in the Americas.
In the 1990’s it reappeared in the former Soviet Union, many victims were adults.  This was because the Public Health that was in place before the break up was gone, as many adult victims as children.  Only 68% of Russian children had received the vaccine.  U.S. vaccination rate during the same period was 90-95%

 

Other Corynebacteria sp.

 

Humans: C. jeikeium,

 C. pseudotuberculosis, C. pseudodiphtheritcum.

Animals:  C.bovis
Plants: C.betae

 

C. jeikeium

 

This species of Corynebacterium is most commonly isolated from human infections. 
This lipophilic species colonizes the skin of hospitalized patients. 
Infections seen in immunocompromised hosts.
This species does cause serious infections. 
First documented Corynebacterium species as being multiresistant to antimicrobial agents.   

 

C. pseudodiphtheriticum

 

This may be an important emerging pathogen in immunocompromised hosts.
Considered normal human oropharyngeal flora.
However it is isolated in respiratory tract infections.  

 

C. bovis

 

This species is part of the normal flora of the bovine udder. 
Rarely causes human infection.

 

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