Corynebacterium diphtheriae: Diphtheria Toxin, Pseudomembrane, Diagnosis, Treatment

A 6-year-old who never completed her childhood vaccinations is brought in with a sore throat, low fever, and a swollen neck. Looking into her throat, the doctor sees something that a simple sore throat never produces: a thick, gray, sheet-like membrane stuck firmly over the tonsils. When a swab is used to lift its edge, the surface underneath bleeds.

The child is not just fighting a throat infection. A bacterium growing on that membrane is releasing a toxin that is already traveling to her heart and nerves. The membrane itself can grow into the airway and block it. This is diphtheria, caused by Corynebacterium diphtheriae, and the priority is not the culture result but neutralizing the toxin without delay.

Corynebacterium diphtheriae, also known as the Klebs-Löffler bacillus, is a gram-positive rod that causes diphtheria. Once a major killer of children, diphtheria became rare after widespread vaccination, but it re-emerges wherever immunization coverage falls. C. diphtheriae barely invades tissue at all, and nearly all the damage, the throat membrane, the heart failure, the nerve palsies, comes from a diphtheria toxin. Once you understand what that toxin does, the clinical picture, the diagnosis, and the treatment all follow.

Figure: Characteristics Pseudomembrane of C. diphtheriae

The names capture the two most testable features. Korynee is Greek for "club," describing the club-shaped rods seen under the microscope, and diphthera means "leather hide," describing the tough pseudomembrane that is the hallmark of respiratory diphtheria.

General properties

• Gram-positive rods that are club-shaped, because of metachromatic (volutin) granules stored at one or both ends. After dividing, the cells snap into sharp angles and stay partly attached, so they line up as parallel "palisades" or as V and L shapes, the "Chinese letter" arrangement. This distinctive arrangement and the granules are what let you suspect C. diphtheriae on a stained smear, though they do not by themselves prove the organism is toxigenic.
• Nonmotile, non-capsulated, and non-sporing.
• Aerobes or facultative anaerobes,
• Fastidious organisms; grows best at 37°C on blood or serum-containing media such as Loeffler’s medium, tellurite medium, etc.
• Ferment glucose and glycogen.
• Catalase positive, non-pigmented, oxidase negative, indole negative, and do not form phosphatase.

Figure: Albert Staining of Corynebacterium diphtheriae

Pathogenesis

Disease Transmission

The source of infection is carriers who harbor the organisms in the oropharynx or skin. Human to human transmission is spread by respiratory droplets, secretions, or direct contact with infected cutaneous lesions.

C. diphtheriae is not invasive. It stays on the surface of the throat or skin. Almost the entire disease is caused by one potent exotoxin, diphtheria toxin. A crucial point for exams and for understanding the organism: only strains infected by a specific bacteriophage (a virus that carries the tox gene) can make the toxin. The toxin gene belongs to the phage, not the bacterium itself. This is why a C. diphtheriae isolate is not automatically dangerous, and why the laboratory must prove toxin production, not just identify the organism.

Corynebacterium diphtheriae is not the part of the normal flora of humans but coryneforms bacteria are normal flora and may mistaken for C. diphtheriae in gram staining)

Mechanism of Diphtheria toxin

Figure: Mechanism of Diphtheria toxin (Image source: Ref-3)

Diphtheria toxin is a classic A-B toxin. The B (binding) subunit attaches the toxin to receptors on the host cell, and the A (active) subunit is the enzyme that does the damage once it is inside.

The mechanism explains the whole disease:

1. The B subunit binds the host cell and the toxin is taken in.
2. The A subunit enters the cytoplasm and acts as an enzyme. It catalyzes ADP-ribosylation of elongation factor 2 (EF-2), a protein the cell needs to build proteins.
3. ADP-ribosylation switches EF-2 off. With EF-2 disabled, the cell can no longer make proteins.
4. Without protein synthesis, the cell dies.

Two features make this toxin especially dangerous. It is extremely potent: a single molecule of the A subunit can inactivate enough EF-2 to kill the cell, because the A subunit works catalytically (it is not used up). And it acts on all cells, so once it spreads through the blood it damages distant organs, especially the heart and nerves.

Putting it together: how the toxin builds the disease

At the throat, toxin kills the surface epithelial cells. The dead cells, together with fibrin, inflammatory cells, and bacteria, pile up into the thick, adherent gray pseudomembrane. Because the membrane is stuck to living tissue underneath, trying to peel it off makes the raw surface bleed, and if the membrane extends into the larynx it can obstruct the airway. Toxin that reaches the bloodstream then damages the heart muscle (myocarditis) and the nerves (palsies), which are the dangerous late complications. Every major feature of diphtheria traces back to this one toxin.

In addition to C. diphtheriae, two other corynebacteria species can produce diphtheria toxin and thus also cause diphtheria: C. ulcerans and very rarely C. pseudotuberculosis.

Clinical manifestations

There are two types of clinical diphtheria: nasopharyngeal (respiratory) and cutaneous.

Respiratory diphtheria

Respiratory diphtheria begins with non-specific fever, sore throat, and swollen neck glands. The specific sign is the thick, gray, adherent pseudomembrane over the tonsils and throat. Two dangers follow, and both come from the toxin. Locally, the membrane can extend into the larynx and block the airway, causing suffocation. A markedly swollen neck ("bull neck") signals severe disease.

Systemically, toxin carried in the blood causes myocarditis (which can be fatal) and neurological damage, classically palsy of the palate and later of other cranial and peripheral nerves. These complications can appear even after the throat looks better, which is why neutralizing the toxin early matters so much.

Cutaneous diphtheria

Cutaneous diphtheria occurs by skin contact with other infected persons and appears as ulcerating skin lesions covered by a gray membrane. These lesions do not invade surrounding tissues and the systemic symptoms rarely occur.

Lab Diagnosis

Laboratory diagnosis has two distinct jobs: first, identify the organism as C. diphtheriae, and second, prove that the isolate actually produces the toxin. Identifying the organism is not enough, because non-toxigenic strains exist and do not cause classic diphtheria. This is why every step below leads toward the toxigenicity test as the definitive answer.

One point that must sit above all the laboratory detail: do not wait for the laboratory to start treatment. Diphtheria is treated on clinical suspicion, because antitoxin only works before the toxin binds tissue (explained under treatment). The laboratory confirms the diagnosis; it does not gate the decision to treat.

Sample: Swabs (preferably two) from the lesion of throat, larynx or nasal cavity; one for direct examination and another for culture or a portion of the pseudomembrane.

Direct examination

Figure: Club shaped Corynebacterium diphtheriae in Methylene Blue Staining Source: Source: PHIL Photo ID# 7323

Throat-swab smears are stained with a Gram stain and with methylene blue or Albert stain. Irregularly stained, tapered, pleomorphic gram-positive rods in Chinese-letter arrangement suggest C. diphtheriae, and the metachromatic (volutin) granules stand out on methylene blue or Albert stain. In Albert stain, the bacilli appear green with bluish-black granules. Remember, the smear supports a clinical suspicion quickly, but it cannot distinguish toxigenic from non-toxigenic strains, and harmless coryneform "diphtheroids" can look similar. For the full method, see the article on the Albert stain.

Culture

Corynebacterium diphtheriae is a fastidious organism, so it does not grow on the ordinary medium. To avoid the growth of commensals and to differentiate among various biotypes, the sample should be cultured on a selective and differential medium such as cystine-tellurite blood agar and modified Tinsdale’s medium.

Figure: Colonies of Corynebacterium diphtheriae biotype gravis in tellurite blood agar (Image source: CDC/ Dr. W.A. Clark )

In addition, Loeffler’s medium, containing serum and eggs, stimulates the growth of C. diphtheriae and stimulates the production of metachromatic granules within the cells. Primary plating in Loeffler’s medium is not recommended due to the growth of commensals.

Blood tellurite agar: It is a selective and differential medium for C. diphtheriae. After 48-72 hours, colonies of C. diphtheriae appear as small, grey, or black with a raised center. Biotypes (gravis, intermedius, mitis, and belfanti) can be differentiated on the basis of colony morphology on tellurite blood agar and biochemical tests such as;

• Urease
• Nitrate reduction
• Esculin hydrolysis
• Fermentation of Glycogen
• Lipophilic characteristics

Figure: Colonies of C. diphtheriae in Tinsdale Agar

Tinsdale’s Agar: After incubation for at least 48 hours, colonies of Corynebacterium diphtheriae appear black with dark brown halos.

Rapid identification methods

API Coryne strip and RapID CB Plus are commercial products available for the rapid identification of Corynebacterium diphtheriae.

Virulence test

This is the decisive step. An isolate is only a true diphtheria pathogen if it produces toxin, so toxigenicity must be demonstrated. The available tests:

• Elek immunodiffusion test: It is the most common in vitro assay for determining toxigenicity of C. diphtheriae. This test is based on the double diffusion of diphtheria toxin and antitoxin in an agar medium. A sterile, antitoxin-saturated filter paper strip is embedded in the culture medium, and C diphtheriae isolates are streak-inoculated at a 90° angle to the filter paper.
  
  The production of diphtheria toxin is shown within 18 to 48 hours by lines of precipitation where toxin from a toxigenic strain meets the antitoxin. A toxigenic isolate produces these precipitin lines; a non-toxigenic one does not. For the full method and how to read it, see the article on the Elek test.
• Detection of toxin gene by polymerase chain reaction (PCR). PCR detects the toxin gene quickly, but carrying the gene is not identical to actively expressing toxin, so PCR is often paired with the Elek test, which shows that functional toxin is actually made.
• Guinea pig lethality test

Treatment of diphtheria

Treatment has two parts, and their order matters.

1. Diphtheria antitoxin (the priority). The single most important treatment is diphtheria antitoxin, and it must be given as early as possible on clinical suspicion, without waiting for laboratory confirmation. The reason follows directly from the mechanism: antitoxin can only neutralize toxin that is still free in the blood. Once the toxin has entered and bound host cells, antitoxin cannot pull it back out. So every hour of delay lets more toxin bind irreversibly to the heart and nerves. This is why diphtheria is a "treat on suspicion" disease. The antitoxin is an animal-derived (equine) product, so it is given with care for hypersensitivity.

2. Antibiotics (supportive). Penicillin or a macrolide (such as erythromycin or azithromycin) is given to kill the organism and stop further toxin production, to help clear the pseudomembrane, and to reduce spread to others. But antibiotics do nothing to the toxin already released, so they support the antitoxin rather than replace it.

Supportive care includes protecting the airway (the membrane can obstruct breathing) and monitoring for myocarditis. Close contacts and carriers are given antibiotics and their immunization is checked.

Prevention and immunization

Prevention is by active immunization with diphtheria toxoid, which is diphtheria toxin inactivated with formaldehyde. The toxoid triggers antibodies that neutralize the toxin, so it protects against the effects of the toxin rather than against carriage of the organism. It is given in childhood in combination with tetanus and pertussis vaccines (as DTP/DTaP), with boosters, because immunity fades over time. This is the same "toxin is the disease, so vaccinate against the toxin" logic seen in tetanus.

How to Remember

Key exam facts

Where Students Get Confused

References and further readings

• Tille, P. M. (2022). Bailey & Scott's Diagnostic Microbiology (15th ed.). Elsevier.
• Procop, G. W., & Koneman, E. W. (2017). Koneman's Color Atlas and Textbook of Diagnostic Microbiology (7th ed.). Wolters Kluwer.
• Carroll, K. C., Pfaller, M. A., et al. (2020). Murray's Medical Microbiology (9th ed.). Elsevier.
• Jamal, S. B., Tiwari, S., Silva, A., & Azevedo, V. (2017). Pathogenesis of Corynebacterium diphtheriae and available vaccines: an overview. Global Journal of Infectious Diseases and Clinical Research, 3(1), 20–24.
• Centers for Disease Control and Prevention. Diphtheria: clinical information. CDC (current version).