Microbiology
Medical bacteriologyDr. Zainab D. Degaim
Microbiology
Medical bacteriology
Dr. Zainab D. Degaim
Corynebacterium diphtheriae
Morphology and IdentificationCorynebacteria are 0.5–1 μm in diameter and several micrometers long. Characteristically, they have irregular swellings at one end that give them the “club-shaped” appearance. Irregularly distributed within the rod (often near the poles) are granules staining deeply with aniline dyes (metachromatic granules) that give the rod a beaded appearance.
Individual corynebacteria in stained smears tend to lie parallel or at acute angles to one another. True branching is rarely observed in cultures.
C diphtheriae and other corynebacteria grow aerobically on most ordinary laboratory media. On blood agar, the C diphtheriae colonies are small, granular, and gray with irregular edges and may have small zones of hemolysis.
On agar containing potassium tellurite, the colonies are brown to black with a brown-black halo because the tellurite is reduced intracellularly.
Four biotypes of C diphtheriae have been widely recognized and each of them produces the potent exotoxin: gravis, mitis, intermedius, and belfanti. These variants have been classified on the basis of growth characteristics such as colony morphology, biochemical reactions, molecular methods such as ribotyping and severity of disease produced by infection.
Corynebacteria tend to pleomorphism in microscopic and colonial morphology.
When some nontoxigenic diphtheria organisms are infected with bacteriophage from certain toxigenic diphtheria bacilli, the offspring of the exposed bacteria are lysogenic and toxigenic, and this trait is subsequently hereditary.
When toxigenic diphtheria bacilli are serially subcultured in specific antiserum against the temperate phage that they carry, they tend to become nontoxigenic.
Thus, acquisition of phage leads to toxigenicity (lysogenic conversion). The actual production of toxin occurs only when the prophage of the lysogenic C diphtheria becomes induced and lyses the cell. Whereas toxigenicity is under the control of the phage gene, invasiveness is under the control of bacterial genes.
Pathogenesis
The principal human pathogen of the genus Corynebacterium is C diphtheriae, the causative agent of respiratory or cutaneous diphtheria.
C diphtheriae occurs in the respiratory tract, in wounds, or on the skin of infected persons or normal carriers.
It is spread by droplets or by contact to susceptible individuals; the bacilli then grow on mucous membranes or in skin abrasions, and those that are toxigenic start producing toxin.
All toxigenic C diphtheriae are capable of elaborating the same disease-producing exotoxin.
In vitro production of this toxin depends some factors such as the concentration of iron, osmotic pressure, amino acid concentration, pH, and availability of suitable carbon and nitrogen sources. The factors that control toxin production in vivo are not well understood.
Diphtheria toxin
is a heat-labile, single-chain, threedomain polypeptide (62 kDa) that can be lethal in a dose of 0.1 μg/kg body weight. If disulfide bonds are broken, the molecule can be split into two fragments.
Fragment B (38 kDa), which has no independent activity, is functionally divided into a receptor domain and a translocation domain.
The binding of the receptor domain to host cell membrane proteins CD-9 and heparin-binding epidermal growth factor (HB-EGF) triggers the entry of the toxin into the cell through receptor-mediated endocytosis.
Acidification of the translocation domain within a developing endosome leads to creation of a protein channel that facilitates movement of Fragment A into the host cell cytoplasm.
Fragment A inhibits polypeptide chain elongation—provided nicotinamide adenine dinucleotide (NAD) is present—by inactivating the elongation factor EF-2. This factor is required for translocation of polypeptidyl-transfer RNA from the acceptor to the donor site on the eukaryotic ribosome.
Toxin Fragment A inactivates EF-2 that the abrupt arrest of protein synthesis is responsible for the necrotizing and neurotoxic effects of diphtheria toxin. An exotoxin with a similar mode of action can be produced by strains of Pseudomonas aeruginosa.
Pathology
Diphtheria toxin is absorbed into the mucous membranes and causes destruction of epithelium and a superficial inflammatory response.
The necrotic epithelium becomes embedded in exuding fibrin and red and white cells, so that a grayish “pseudomembrane” is formed—commonly over the tonsils, pharynx, or larynx.
The regional lymph nodes in the neck enlarge, and there may be marked edema of the entire neck, with distortion of the airway, often referred to as “bull neck” clinically.
The diphtheria bacilli within the membrane continue to produce toxin actively. This is absorbed and results in distant toxic damage, mostly parenchymatous degeneration, fatty infiltration, and necrosis in heart muscle (myocarditis), liver, kidneys (tubular necrosis), and adrenal glands, sometimes accompanied by gross hemorrhage.
The toxin also produces nerve damage (demyelination), often resulting in paralysis of the soft palate, eye muscles.
Wound or skin diphtheria occurs chiefly in the tropics, although cases have also been described in temperate climates among alcoholic, homeless individuals, and other poor groups. A membrane may form on an infected wound that fails to heal. However, absorption of toxin is usually small and the systemic effects negligible. The small amount of toxin that is absorbed during skin infection promotes development of antitoxin antibodies.
C diphtheriae does not typically invade deep tissues and practically never enters the bloodstream. However, notably over the last two decades, reports of invasive infections such as endocarditis and septicemia due to non-toxigenic C diphtheria have increased.
Clinical Findings
When diphtheritic inflammation begins in the respiratory tract, sore throat and low-grade fever usually develop.
Prostration and dyspnea soon follow because of the obstruction caused by the membrane. This obstruction may even cause suffocation if not promptly relieved by intubation or tracheostomy.
Irregularities of cardiac rhythm indicate damage to the heart.
Later, there may be difficulties with vision, speech, swallowing, or movement of the arms or legs.
In general, var gravis tends to produce more severe disease than var mitis, but similar illness can be produced by all types.
Diagnostic Laboratory Tests
Swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered.
The swab should then be placed in semisolid transport media such as Amies. Smears stained with alkaline methylene blue or Gram stain show beaded rods in typical arrangement.
Specimens should be inoculated to a blood agar plate (to rule out hemolytic streptococci) and a selective medium such as a tellurite plate (eg, cystine-tellurite blood agar [CTBA]) and incubated at 37°C in 5% CO2. Plates should be examined in 18–24 hours. In 36–48 hours, the colonies on tellurite medium are sufficiently definite for recognition of C diphtheriae. On cystine tellurite agar, the colonies are black with a brown halo.
A presumptive C diphtheriae isolate should be subjected to testing for toxigenicity. Such tests are performed only in reference public health laboratories. There are several methods, as follows:
1. Modified Elek immunoprecipitation method. A filter paper disk containing antitoxin (10 IU/disk) is placed on an agar plate which contain at least 10 colonies should be chosen) for toxigenicity are spot inoculated 7–9 mm away from the disk. After 48 hours of incubation, the antitoxin diffusing from the paper disk has precipitated the toxin diffusing from toxigenic cultures and has resulted in precipitin bands between the disk and the bacterial growth.
2. Polymerase chain reaction (PCR) for detection of the diphtheria toxin gene (tox). PCR assays for tox can also be used directly on patient specimens before culture results are available.
A positive culture result confirms a positive PCR assay. A negative culture result after antibiotic therapy along with a positive PCR assay result suggests that the patient probably has diphtheria.
3. Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C diphtheria isolates.
4. An immunochromatographic strip assay allows detection of diphtheria toxin in a matter of hours. This assay is highly sensitive. The latter two assays are not widely available.
Resistance and Immunity
Because diphtheria is principally the result of the action of the toxin formed by the organism rather than invasion by the organism, resistance to the disease depends largely on the availability of specific neutralizing antitoxin in the bloodstream and tissues.
It is generally true that diphtheria occurs only in persons who possess no antitoxin antibodies (IgG) (or less than 0.1 IU/mL). Assessment of immunity to diphtheria toxin for individual patients can best be made by review of documented diphtheria toxoid immunizations and primary or booster immunization if needed.
Treatment
The treatment of diphtheria rests largely on rapid suppression of toxin-producing bacteria by antimicrobial drugs and the early administration of specific antitoxin against the toxin formed by the organisms at their site of entry and multiplication.
Diphtheria antitoxin is produced in various animals (horses, sheep, goats, and rabbits) by the repeated injection of purified and concentrated toxoid. From 20,000 to 120,000 units are injected intramuscularly or intravenously depending on the duration of symptoms and severity of illness after suitable precautions have been taken (skin test) to rule out hypersensitivity to the animal serum.
The antitoxin should be given intravenously on the day the clinical diagnosis of diphtheria is made and need not be repeated. Intramuscular injection may be used in mild cases. Diphtheria antitoxin will only neutralize circulating toxin that is not bound to tissue.
Antimicrobial drugs (penicillin, macrolides) inhibit the growth of diphtheria bacilli. Although these drugs have virtually no effect on the disease process, they arrest toxin production and assist public health efforts. Antimicrobial resistance to these agents is rare.