The History of Discovery


  • Causative agent of diphtheria, Corynebacterium diphtheria, was discovered by E. Klebs in 1883. F. Loeffler isolated it in pure culture in 1884. E. Roux and A. Yersin first derived the main virulence factor of Corynebacterium diphtheria, diphtheria exotoxin, in 1888. Corresponding antitoxin antibodies were obtained by E. Behring and S. Kitasato in 1890. Finally, G. Ramon created first biological product for specific prophylaxis of diphtheria, diphtheria toxoid, in 1923.

Classification


Corynebacterium diphtheria pertains to family Corynebacteriaceae, genus Corynebacterium. It accounts for an extremely dangerous toxinemic infection – diphtheria. Closely related species Corynebacterium ulcerans and Corynebacterium pseudotuberculosis can carry tox gene that encodes diphtheria toxin production, thereby they can also exert the disease in rare conditions.


Structure and Properties of Corynebacterium diphtheria


Morphology

  • Corynebacterium diphtheriae (Lat. coryna – club) is a straight or slightly curved polymorphic rod 1-8 μm in length. Under microscopy the groups of bacteria resemble letters X or V. Branched and thread-like forms as well as short coccobacterial forms may occur.
  • The rods of C. diphtheriae frequently display terminal club-shaped bulges with volutin granules, stained blue by Neisser stain. Volutin is the store of polyphosphates for microbial cells.
  • Granules of volutin are detected also by luminescent microscopy (e.g., they stain orange-red with coriphosphine dye).
  • Microbial cells are gram-positive. They have no spores or flagella, but may possess capsule.

Cultivation

  • The optimal temperature for microbial growth is about 37°С, and the bacteria can’t propagate at temperatures below 15°С and above 45°С. Optimal medium pH is 7.2-7.6.
  • These organisms grow in media enriched with proteins (coagulated serum, blood agar, and serum agar) or in sugar broth. On Roux (coagulated horse serum) or Loeffler medium (three parts of bovine serum and one part of sugar broth) visible growth appear in 16-18 hours.
  • Now blood tellurite agar, containing blood and potassium tellurite (Clauberg II medium) and cystine-tellurite agar or Tinsdale medium are most often used for C. diphtheriae culture.
  • According to cultural and biological properties, various biovars of C. diphtheriae were defined: gravis, mitis, and intermedius, which differ in a number of properties. Recently a new C. diphtheriae biovar belfanti was described.
  • Corynebacteria of the gravis biovar produce large rough (R forms) rosette-like black or grey colonies on tellurite agar. The bacteria ferment starch with acid end products and produce a pellicle in meat broth. They are usually highly toxic with marked invasive properties.
  • The colonies produced by corynebacteria of mitis biovar on tellurite agar are dark, smooth (S forms), and glistening. Starch is not fermented. Bacteria cause hemolysis of animal erythrocytes and produce diffuse turbidity in meat broth. Cultures of this biovar are usually less toxic and invasive than those of gravis biovar.
  • The bacteria of intermedius biovar are transitional. They produce small (R-S forms) black colonies on tellurite agar. Starch is not fermented. Growth in meat broth results in turbidity.
  • Newly discovered biovar belfanti is similar to other bacteria, but can’t reduce nitrates into nitrites.
  • It was proven that gravis biovar is isolated in epidemic outbreaks of diphtheria, while mitis biovar appears in sporadic cases of the disease.

Biochemical properties

  • The causative agent of diphtheria is facultatively anaerobic bacterium. C. diphtheriae ferments glucose with acid formation, whereas galactose, maltose, starch, and glycerol fermentation is variable.
  • Bacteria have no urease, produce no indole, and slowly produce hydrogen sulfide. They reduce nitrates to nitrites except biovar belfanti.
  • C. diphtheriae has the enzyme cystinase that is determined in Pizu test (serum agar media with cystine and lead acetate is blackened due to lead sulfide production). Conversely, diphtheria agents have no pyrazinamidase enzyme.
  • Production of cystinase and lack of pyrazinamidase distinguishes C. diphtheriae from other corynebacteria.
  • C. diphtheriae expresses the number of virulence enzymes – catalase, hyaluronidase, neuraminidase, and DNAse.

Antigenic structure

  • There are two major antigenic fractions in corynebacteria. Superficial heat-labile type-specific K-antigen is of protein nature.
  • Somatic group-specific lipopolysaccharide O-Ag is heat stable.
  • To date 57 serotypes of C. diphtheriae have been determined by agglutination reaction.

Virulence factors

  • All toxigenic С. diphtheriae express extremely poisonous exotoxin.
  • Bacterial toxigenicity is under the control of phage genes. When some nontoxigenic diphtheria strains are infected with bacteriophage transduced from toxigenic diphtheria agent, the offsprings of the exposed bacteria become lysogenic and toxigenic.
  • Thus, acquisition of phage leads to toxigenicity (lysogenic conversion). The actual production of toxin usually occurs only after activation of the prophage within lysogenic С. diphtheriae.
  • In addition, toxin synthesis is governed by transcriptional regulator diphtheria toxin repressor (DtxR) encoded by nucleoid dtxR gene.
  • DtxR is iron-dependent transcriptional regulator. When the concentration of iron is sufficient, it blocks the expression of diphtheria toxin. And vice versa, low iron concentrations render DtxR repressor inactive, allowing the synthesis of exotoxin.
  • Diphtheria toxin is a heat-labile polypeptide with molecular weight 62,000. After inner thiol reduction the molecule is splitted into two fragments.
  • Portion В is required for the transport of fragment A into the cell. Fragment A inhibits peptide chain elongation factor EF-2 by its ADP-ribosylation.
  • Block of protein synthesis disrupts normal cellular functions. Abrupt termination of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin. Pure diphtheria toxin may be lethal in extremely low dose of 40 ng.
  • Other virulence factors include adhesive pili and fimbria, invasive enzymes, hemolysins, and cord-factor.
  • Cord-factor of С. diphtheriae (trehalose dimycolate) damages mitochondria, affecting the processes of respiration and phosphorylation.

Resistance

C. diphtheriae are relatively resistant to various environmental factors. For instance, they survive for two months at room temperature. Corynebacteria remain viable in the membranes of diphtheria patients at least for 2 weeks, in water and milk – for 20 days. The bacteria are killed by a temperature of 60°С and by 1% phenol solution in 10 minutes.


Pathogenesis and Clinical Findings in Diphtheria


  • Patients suffering from the disease and carriers are the main sources of infection in diphtheria.
  • The disease is communicated by airborne (air droplet or air-dust) route. Transmission by various objects or fomites (toys, books, towels, utensils, etc.) and foodstuffs (e.g., milk) contaminated with C. diphtheriae is also possible.
  • Exotoxin plays the principal role in the pathogenesis of diphtheria, blocking protein synthesis.
  • It crosses the mucous membranes and causes the destruction of epithelium. The necrotic epithelium forms grayish “pseudomembranes” over the tonsils, pharynx, or larynx. They are tightly bound to the affected tissues.
  • Any attempt to remove the pseudomembrane results in bleeding. Pseudomembrane respiratory obstruction (diphtheritic croup) can cause patient suffocation.
  • The regional lymph nodes in the neck enlarge, and there may be total neck edema. The diphtheria agents continue to produce toxin within the membranes.
  • Toxin absorption results in distant toxic action with tissue damage, particularly degeneration and necrosis in myocardium, liver, kidneys, and adrenals, sometimes accompanied by hemorrhages.
  • The toxin also exerts nerve damage, resulting often in paralysis of the soft palate, eye muscles, or limbs.
  • The incidence of diphtheria of other organs (eyes, ears, skin or genital tract) is much seldom.
  • Post-infectious active immunity depends mainly on the antitoxin contents in the blood. However, a definite role of the antibacterial immunity, associated with phagocytosis, T cells, opsonization and complement-dependent microbial lysis is also significant. Therefore, the immune response produced by diphtheria infection is both antitoxic and antibacterial.
  • In general, diphtheria confers not very stable immunity, thus reinfection may occur up to 10% of cases.

Laboratory Diagnosis of Diphtheria


  • Swabs from the throat, nose, or other lesions as well as diphtheria pseudomembranes are tested as clinical specimens.
  • Neisser-stained smears are examined and reveal typical corynebacteria with volutin granules. As rapid sensitive test, luminescent microscopy is used with coriphosphine staining that determines the presence of orange-stained volutin granules within microbial cells.
  • Nonetheless, diphtheria diagnosis is confirmed only in case of exotoxin detection in the clinical specimen or in isolated culture.
  • Rapid determination of diphtheria exotoxin in clinical samples is elaborated by ELISA; identification of microbial tox-genes is performed by PCR.
  • Overall, PCR is regarded as the most sensitive, rapid and specific test for the confirmation of toxigenicity of С. diphtheriae.
  • When cultured, the specimens are planted onto special media, e.g. Loeffler coagulated serum, Clauberg II medium, Tinsdale agar, etc.
  • Primary growth is assessed on the Loeffler slant in 12-18 hours. In 36-48 hours the typical colonies on tellurite-containing media are observed.
  • The isolated culture is further identified by biochemical and antigenic tests and by phage typing.
  • For the determination of toxigenicity of isolated cultures various neutralization tests are applied.
  • In case of animal experimental infection, the material can be injected into 2 groups of animals (guinea pigs or mice), where one of them was passively protected with diphtheria antitoxin. The unprotected animals die in 2-3 days, whereas the immunized ones survive.
  • Plate immunoprecipitation or Elek’s test is made as follows: a strip of filter paper saturated with antitoxin is placed onto serum agar plate. The cultures to be tested for toxigenicity are streaked across the plate at right angles to the filter paper.
  • After 16-24 hour incubation the antitoxin diffusing from the paper strip yields the precipitation of toxin diffusing from toxigenic cultures. As the result, precipitation lines are determined between the strip and bacterial growth.
  • The toxigenicity of С. diphtheriae can be also shown by inoculation of bacteria into cell culture monolayers (e.g., Vero cell cultures). It is followed by evidenr cytopathic effect of the toxin with the destruction of cell monolayer.

Specific Treatment and Prophylaxis of Diphtheria


  • The specific treatment of diphtheria rests largely on the early administration of specific antitoxic antibodies that neutralize highly poisonous exotoxin of С. diphtheriae. Treatment with antibiotics that causes rapid suppression of toxin-producing bacteria is also helpful in the disease management.
  • Diphtheria antitoxin (DAT) is horse serum-derived biological product. It is obtained by the repeated immunizations of horses with purified and concentrated toxoid with subsequent purification.
  • Treatment with antitoxin is mandatory for patient’s recovery. From 20,000 to 100,000 units are injected depending on disease severety.
  • Skin test should be made before antitoxin treatment to detect possible hypersensitivity to animal serum proteins.
  • Antimicrobial drugs (e.g., penicillin G, clarithromycin or azithromycin) inhibit the growth of diphtheria agents. As the result, they greatly diminish toxin production. Antibiotics also help to eliminate coexistent pathogenic bacteria (e.g., streptococci) from the respiratory tracts of affected patients.
  • Specific prophylaxis is achieved by active immunization. Usually DPT vaccine or combined tetanus-diphtheria toxoid are used.
  • It should be emphasized that diphtheria is regarded as the disease fully preventable by vaccination.
  • Population (or “herd”) immunity above 95% is regarded as sufficient to cease the disease contraction among the individuals.
  • All the children must receive the course of diphtheria toxoid immunization. It is afforded thrice at the first year of life starting from the age of 3 month. Subsequent boosters are injected in 9-12 months and then reproduced every 10 years.