BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

Corynebacterium Summary

 
 
Gram stain of Corynebacterium spp. demonstrating "Chinese letters" formations

General Overview:

  C. diphtheriae and related organisms are collectively termed coryneforms or diphtheroids

  Corynebacteria possess capsular (K) and somatic antigens (O)

Morphology & Physiology:

  Small, nonmotile, irregularly staining pleomorphic Gram-positive rods with club-shaped swelled ends but no spores; may be straight or slightly curved (see WebLinked image; see WebLinked image)

  Palisade arrangement of cells in short chains ("V" or "Y" configurations) or in clumps resembling "Chinese letters"

  Cells tend to lie parallel to one another (palisades) or at acute angles (coryneforms), due to their snapping type of division
  Vary greatly in dimension, from 0.3 to 1 um in diameter and 1.0 to 8.0 um in length

  May also contain inclusion bodies, known as metachromatic granules, which are composed of inorganic polyphosphates (volutin) that serve as energy reserves and are not membrane bound

   Internal metachromatic granules densely stain ruby red while the rest of the bacillus stains blue, when stained with an aniline dye such as toluidine blue O or methylene blue
   Cells appear to be banded or beaded with irregularly staining granules; may show alternate bands of stained and unstained material (giving the appearance of septa)

  Aerobic or facultatively anaerobic

  Fermentative metabolism (carbohydrates to lactic acid); form acid but not gas from certain carbohydrates

  Fastidious; Slow growth on enriched medium

  Catalase positive

  Cell wall containing unusual lipids: meso-diaminopimelic acids; arabino-galactan polymers; short-chain mycolic acids (member of CMN (Corynebacterium, Mycobacterium, Nocardia) group)

  Corynebacterium urealyticum strongly urease positive

Clinical Syndromes:

  Determined by site of infection, host immunity, and virulence of the organism

  Corynebacterium diptheriae: toxigenic strains cause diphtheria in humans

   Respiratory disease

  Initially: sore throat, low-grade fever; followed by adherent pseudomembrane on the tonsils and pharynx

  Later stages include localized damage, bleeding, difficulty in breathing, and myocarditis and peripheral neuritis

  Complications from systemic spread of exotoxin to other target organs in the body; eg., heart (concept of "disease at a distance")

  Most mortality from systemic toxin-mediated heart failure

   Cutaneous diphtheria (extra-respiratory disease)

  Acquired by skin contact; organism enters through break in subcutaneous tissue

  Chronic non-healing ulcer results

  Corynebacterium jeikeium: opportunistic infections (especially in immunocompromised patients)

  Corynebacterium urealyticum: urinary tract infections (UTI’s); rare but important

  Corynebacterium pseudotuberculosis: subacute relapsing lymphadenitis

  Corynebacterium ulcerans: pharnygitis

  Corynebacterium xerosis: bacteremia, skin infections, pneumonia in immunocompromised hosts (e.g., patients with blood disorders, bone marrow transplants, intravenous catheters) and pharyngitis

  Corynebacterium pseudodiphtheriticum: endocarditis and lower-respiratory tract infections

Epidemiology:

  Widely distributed in nature; worldwide in occurrence

   Only 28 cases reported between 1980-1990 in the U. S. due to highly successful immunization program
   More commonly occurring in other countries
   Former Soviet States have had epidemic rise in incidence since breakup and disruption of immunization program

  Human is the only natural host

  Corynebacterium diptheriae:

  Diphtheria (respiratory or cutaneous) occurs worldwide primarily in urban areas
  Carried assymptomicatically in the oropharynx of immune individuals
  Transmitted by respiratory droplets or skin contact

  Corynebacterium jeikeium: carriage on skin of up to 40% of hospitalized patients (e.g., bone marrow transplants)

  Several species form part of the common microbiota of the human respiratory tract and other mucous membranes, the conjunctiva, and the skin

Pathogenesis & Immunity:

  Non-pathogenic species are called "diphtheroids"; two species commonly found in humans are Corynebacterium xerosis and Corynebacterium pseudodiphtheriticum

  Pathogenic type species is Corynebacterium diphtheriae, which produces a potent exotoxin and causes diphtheria in humans

   Diptheria A-B exotoxin interrupts peptide formation at the ribosomal level
   Phospholipase D increases vascular permeability, thus allowing C. diphtheriae to spread through tissues of the naso-pharyngeal area
   Toxin Characteristics:

  Encoded by tox gene introduced by lysogenic bacteriophage (prophage) in virulent strains of C. diphtheriae

  63,000 dalton protein toxin consisting of two fragments, A and B

  Prototype A-B exotoxin acts systemically

1. B fragment binds to receptor sites on target cells and toxin is internalized by receptor-mediated endocytosis
2. A fragment blocks protein synthesis by ADP-ribosylation of elongation factor-2 (EF-2)

  Produced in the presence of limiting amounts of iron; optimum toxin production in vitro occurs in the presence of 100 mg iron per liter

  Used to produce toxoid in DPT and TD vaccines (see below)

  Corynebacterium jeikeium: multiple antibiotic resistance important in opportunistic infections of immunocompromised patients

  Corynebacterium urealyticum: urease hydrolyzes urea; release of NH₄⁺, increase in pH, alkaline urine, renal stones

Laboratory Identification:

  Microscopy

  Methylene blue stain shows metachromatic granules
  Gram stain shows Gram-positive pleomorphic rods arranged in perpendicular, parallel, and pallisade formations

  Culture

   A confirmed diagnosis of diphtheria can only be made by isolating toxigenic diphtheria bacilli from the primary lesion (in the throat or elsewhere)
   Exudate from the lesion should be inoculated onto blood agar and selective media: cysteine-tellurite agar; serum tellurite agar; Loeffler’s slant:

  Three varieties of C. diphtheriae colonies may be recognized: gravis, intermedius, and mitis colonial morphology:

1. var. gravis: large, flat, rough, dark-gray colonies; not hemolytic; very few small metachromatic granules; form a pellicle in broth
   
2. var. mitis: smooth, convex, black, shiny, entire colonies; hemolytic; prominent metachromatic granules; diffuse turbidity in broth
   
3. var. intermedius: dwarf, flat, umbilicate colony with a black center and slightly crenated periphery; not hemolytic; fine granular deposit in broth

  C. diphtheriae (also Staphylococcus) produces gray to black colonies on the tellurite media because the tellurite is reduced intracellularly to tellurium

  Any colonies which appear on the three media should be stained with toluidine blue O or methylene blue

  Any typical Corynebacterium colonies would be subcultured on a Loeffler's slant, and tested for toxigenicity, either by the guinea pig virulence test or by the in vitro gel diffusion method of Elek

  Diphtheroids may be distinguished from C. diphtheriae by means of CTA sugar fermentation reactions and tests for toxigenicity

  In vivo test:

   Schick test
   Guinea pig virulence test

  In vitro test: Elek test (immunodiffusion)

Treatment, Prevention & Control:

  Antitoxin

  Used for neutralizing exotoxin
  Effective in conjunction with antibiotic therapy

  Toxoid

  Toxoid preparations are used for vaccines as active immunization for diphtheria
  Usually given in conjunction with pertussis and tetanus vaccines (DPT vaccine) or as a booster with tetanus (TD)

  Antibiotics

  Penicillin G
  Erythromycin if allergic

Distinguishing Characteristics of Corynebacterium spp.

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