Corynebacterium diphtheriae (Klebs-Löffler bacillus)- An Overview

Corynebacterium diphtheriae is a gram-positive, rod-shaped bacterium that can cause diphtheria, a contagious infection that affects the throat and sometimes the skin . The bacterium produces a toxin that inhibits protein synthesis in human cells and can damage the nervous system . The toxin is encoded by a virus that infects the bacterium. The bacterium has four biovars based on colonial morphology and biochemical profiles. It is non-motile, non-encapsulated, and non-sporulating . It belongs to the order Actinomycetales, which are typically found in soil.

Diphtheria is a serious infection that can lead to difficulty breathing, heart rhythm problems, and even death. It is spread by droplets or by contact with infected persons or carriers . The bacteria multiply on the mucous membranes of the respiratory tract or on the skin and produce toxin that damages the local tissues and spreads through the bloodstream . The toxin can affect various organs, especially the heart and the nervous system . Diphtheria can be prevented by vaccination with diphtheria toxoid, which induces antibodies that neutralize the toxin .

Corynebacterium diphtheriae is a medically important bacterium that requires prompt diagnosis and treatment. It is also a model organism for studying bacterial pathogenesis and gene regulation by bacteriophages. In this article, we will review the habitat, morphology, genome, cultural characteristics, pathogenesis, clinical manifestations, laboratory diagnosis, treatment, and prevention of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is a bacterium that can be found in various natural environments, such as soil, water, plants, and food products . However, it is mainly associated with the human host, where it can colonize the mucous membranes of the respiratory tract and the skin .

The bacterium can be transmitted from person to person through respiratory droplets or direct contact with infected lesions . It can also be acquired from contaminated objects or fomites. Humans are the only known reservoir for the disease-causing strains of C. diphtheriae, although some toxigenic strains have been isolated from horses.

C. diphtheriae can persist in the nasopharynx or skin of asymptomatic carriers, who can serve as a source of infection for susceptible individuals . The carriage rate of C. diphtheriae varies depending on the geographic region, immunization status, and socioeconomic factors of the population. Carriers can harbor the bacterium for up to three months after infection.

C. diphtheriae is not a highly invasive organism and usually remains localized at the site of entry. However, it can produce a potent exotoxin that can cause systemic damage to various organs and tissues . The production of the exotoxin is regulated by several factors, such as iron concentration, pH, amino acids, and osmolarity of the medium. The exotoxin gene is carried by a lysogenic bacteriophage that can infect different strains of C. diphtheriae and convert them into toxigenic strains .

C. diphtheriae is a mesophilic bacterium that grows optimally at 37°C in aerobic or facultatively anaerobic conditions . It requires nicotinic acid and pantothenic acid for growth, and some strains also need thiamine, biotin, or pimelic acid. It can grow on simple nutrient agar or agar containing blood, but not on enteric agar or MacConkey agar. It can also grow on selective media that contain compounds that inhibit gram-negative bacteria, such as nalidixic acid or colistin sulfate. The most commonly used selective media for C. diphtheriae are cystine-tellurite blood agar (CTBA) and Tinsdale medium .

Corynebacterium diphtheriae is a rod-shaped, Gram-positive bacterium that does not form spores or capsules. It has a characteristic club-shaped appearance due to irregular swelling at one or both ends. It is also thin and slender, measuring about 3 to 6 micrometers in length and 0.5 to 0.8 micrometers in diameter .

Corynebacterium diphtheriae is non-motile and often shows pleomorphism (variation in shape and size) under different conditions. It can be seen in pairs or in small groups, arranged at various angles to each other, forming patterns that resemble Chinese letters, V or L shapes .

One of the distinctive features of Corynebacterium diphtheriae is the presence of metachromatic granules in its cytoplasm . These are accumulations of polyphosphate that serve as energy reserves for the bacterium. They stain bluish-purple with methylene blue, while the rest of the cell stains blue-green. They are usually located at the poles of the cell, giving it a beaded appearance .

The cell wall of Corynebacterium diphtheriae is lipid-rich and contains meso-diaminopimelic acid, arabino-galactan polymers and short-chain mycolic acids. It also has pili (hair-like appendages) that help the bacterium adhere to host cells and surfaces.

Corynebacterium diphtheriae can be visualized by different staining methods, such as Gram stain, methylene blue stain, Albert`s stain and Ponder`s stain . Albert`s stain and Ponder`s stain are specific for detecting metachromatic granules. Albert`s stain produces green cells with bluish-black granules at the poles, while Ponder`s stain produces red cells with purple granules at the poles.

Corynebacterium diphtheriae has a single circular chromosome that is about 2.5 megabases (Mb) in length and has an average G+C content of 53.5% . The genome contains 2,389 genes, of which 2,272 are protein-coding genes and 69 are structural RNA genes. The coding density of the genome is around 88%.

The genome of C. diphtheriae does not contain any plasmids, but it does contain pathogenicity islands (PAIs) that are regions of DNA that encode virulence factors such as the diphtheria toxin. The tox gene that codes for the exotoxin is introduced into strains of C. diphtheriae by a lysogenic bacteriophage, β-phage. The expression of the tox gene is regulated by a chromosomally encoded element, diphtheria toxin repressor (DTxR), which is activated by high iron concentrations and inhibits toxin production.

The genome of C. diphtheriae is highly conserved among different strains and biotypes, but there are also some variations in gene content and arrangement. The core genome of C. diphtheriae consists of around 1,632 genes that are shared by all strains, while the pan-genome consists of around 4,786 genes that include strain-specific genes. Some of these strain-specific genes are involved in antibiotic resistance, iron metabolism, and cell wall biosynthesis.

The genome of C. diphtheriae provides insights into the biology and pathogenicity of this important human pathogen. It also enables the identification of novel targets for antimicrobial and vaccine development.

Corynebacterium diphtheriae grows well on simple nutrient agar and agar containing blood at the mesophilic temperature range. The growth, however, is not observed on enteric agar formulation and on MacConkey agar.

The growth of C. diphtheriae on agar media is accomplished by growing it on media containing compounds like nalidixic acid and colistin sulfate that are inhibitory to gram-negative bacteria.

Like most corynebacteria, C. diphtheriae also grows well at 37 °C or within the mesophilic temperature range.

Some strains of C. diphtheriae are facultative anaerobes that grow well in a CO2-enriched atmosphere.

The most important culture media used for the isolation of C. diphtheriae is the Sheep Blood Agar (SBA). Other selective media include cystine-tellurite blood agar (CTBA) or freshly prepared Tinsdale medium.

The tellurite present in the medium inhibits the growth of other noncorynebacterium species, but some strains of C. diphtheriae are sensitive to potassium tellurite and require the SBA agar.

For the direct culturing of C. diphtheriae, Tinsdale medium is the best medium, but the medium has a short shelf life.

The identification of C. diphtheriae on Tinsdale medium is efficient in the identification of C. diphtheriae as the bacteria shows both tellurite reductase and cystinase activity.

C. diphtheriae belongs to the Hazard group 2 organisms which might require Containment Level 3 conditions for processing in some cases due to aerosol production.

Growth on serum-based media like Loeffler slants produce colonies with pleomorphic rods, some of which might be club-shaped. The cells might have a beaded appearance due to the terminal reddish-purple polyphosphate granules.

Members of C. diphtheriae have been divided into four different biotypes based on their colony morphology on different agar medium. The following are some cultural characteristics of C. diphtheriae on different culture media:

• Tellurite Medium

  • The colony morphology of different biotypes of C. diphtheriae on the tellurite medium might be different.
  • C. diphtheriae biotype biovar gravis forms dull, grey-coloured opaque colonies that have an average diameter of 2 mm. The surface of the colonies is matt, friable that tends to break into small segments when touched with a wire.
  • C. diphtheriae biotype biovar mitis produces opaque grey or black colonies ranging in size between 1.5-2 mm in diameter. The colonies have entire edge and glossy smooth surface. The variation in size is common in this biotype.
  • C. diphtheriae biotype biovar intermedius forms small grey-coloured shiny colonies that are comparatively smaller in size (0.5-1.0 mm in diameter). The colonies are discrete and translucent on the surface.
  • C. diphtheriae biotype biovar belfanti produces grey or black-coloured colonies of 1.5-2.0 mm diameter. The colonies have an entire edge and glossy smooth surface.
  • The black coloured colonies of C. diphtheriae on tellurite agar are due to the tellurite reductase activity of the bacteria. Besides, these can also produce a brown halo around the colonies due to the cystinase activity.

• Sheep Blood Agar

  • Small grey to black-coloured colonies is observed on the sheep blood agar. The colonies usually range between 1-2 mm in diameter.
  • C. diphtheriae exhibits hemolytic activity on blood agar, which might differ in different biotypes.
  • C. diphtheriae biotype biovar gravis is non-hemolytic and thus doesn’t cause clearing of the media.
  • The rest three biovars of the species; mitis, intermedius and belfanti produce colonies that exhibit a small zone of β-hemolysis on the agar.

Corynebacterium diphtheriae is a non-invasive bacterium that causes disease by producing a potent exotoxin that affects various tissues and organs. The exotoxin is encoded by a gene (tox) that is carried by a lysogenic bacteriophage (β-phage) that infects some strains of C. diphtheriae . The expression of the tox gene is regulated by iron concentration and other environmental factors.

The transmission of C. diphtheriae occurs through respiratory droplets, secretions, or direct contact with infected persons or carriers . The bacteria colonize the mucous membranes of the nasopharynx or the skin lesions, where they multiply and secrete the exotoxin . The exotoxin causes local damage to the epithelial cells, resulting in inflammation and necrosis . A characteristic feature of diphtheria infection is the formation of a pseudomembrane, which is a thick layer of fibrin, bacteria, and dead cells that covers the affected area . The pseudomembrane can obstruct the airway and cause respiratory distress .

The exotoxin can also enter the bloodstream and reach distant organs, such as the heart, nerves, kidneys, and adrenal glands . The exotoxin is composed of two subunits: A and B . The B subunit binds to a specific receptor (heparin-binding epidermal growth factor) on the surface of susceptible cells . This facilitates the entry of the A subunit into the cytoplasm via endocytosis . The A subunit is an enzyme that catalyzes the ADP-ribosylation of elongation factor-2 (EF-2), which is essential for protein synthesis in eukaryotic cells . By inactivating EF-2, the exotoxin blocks protein synthesis and induces cell death .

The systemic effects of the exotoxin depend on the dose, the duration of exposure, and the susceptibility of the host . The most common complications of diphtheria are myocarditis (inflammation of the heart muscle), neuritis (inflammation of the nerves), and renal failure (impairment of kidney function) . These can lead to arrhythmias, paralysis, edema, and death if not treated promptly .

Corynebacterium diphtheriae can cause two types of infections: respiratory and cutaneous. The clinical manifestations depend on the site of infection, the production of toxin by the bacteria, and the immune status of the host.

Respiratory diphtheria

• Respiratory diphtheria is typically caused by toxin-producing strains of C. diphtheriae that colonize the mucous membranes of the upper respiratory tract.
• The symptoms usually begin 2 to 5 days after exposure and include sore throat, malaise, low-grade fever, and cervical lymphadenopathy .
• The hallmark of respiratory diphtheria is the formation of a pseudomembrane that covers the throat and tonsils. The pseudomembrane is composed of bacteria, dead cells, fibrin, and inflammatory cells and adheres firmly to the underlying tissue .
• The pseudomembrane can cause difficulty breathing, hoarseness, and coughing. It can also extend to the nasal passages, larynx, trachea, or bronchi and cause nasal discharge, stridor, or respiratory distress .
• In severe cases, respiratory diphtheria can lead to a "bull neck" appearance due to massive swelling of the neck and submandibular region .
• The toxin produced by C. diphtheriae can also be absorbed into the bloodstream and cause systemic complications such as myocarditis (inflammation of the heart muscle), neuropathy (damage to the nerves), and nephritis (inflammation of the kidneys) .
• The most common neurological complication is palatal paralysis, which causes difficulty swallowing and nasal regurgitation. Other neurological complications include cranial nerve palsies, peripheral neuropathy, and Guillain-Barré syndrome .
• The most common cardiac complication is arrhythmia (irregular heartbeat), which can be fatal. Other cardiac complications include heart failure, myocardial infarction, and pericarditis .

Cutaneous diphtheria

• Cutaneous diphtheria is caused by C. diphtheriae or other Corynebacterium species that infect the skin through breaks or wounds.
• The infection usually starts as a papule (small raised bump) that evolves into a chronic ulcer with a grayish membrane .
• The ulcer may be surrounded by erythema (redness) and induration (hardening) and may have a foul-smelling discharge .
• Cutaneous diphtheria can also cause systemic symptoms such as fever, malaise, and lymphadenopathy .
• The risk of toxin production and systemic complications is lower in cutaneous diphtheria than in respiratory diphtheria . However, some cases of myocarditis and neuropathy have been reported in patients with cutaneous diphtheria .

The laboratory diagnosis of Corynebacterium diphtheriae involves the following steps:

• Specimen collection and transport
• Microscopic examination
• Culture and isolation
• Toxigenicity testing
• Antibody detection

Specimen collection and transport

• Dacron swabs from the nose, throat, or other suspected lesions must be obtained before antimicrobial drugs are administered .
• Swabs should be collected from beneath any visible membrane.
• The swab should then be placed in semisolid transport media such as Amies.
• Specimens should be transported to the laboratory as soon as possible.

Microscopic examination

• Smears stained with alkaline methylene blue or Gram stain show beaded rods in typical arrangement.
• Cells often contain metachromatic granules (polymetaphosphate), which stain bluish-purple with methylene blue.
• In Gram staining, purple coloured beaded rods, angular and palisade arrangements that create a ‘Chinese character’ effect are observed.

Culture and isolation

• Specimens should be inoculated to a blood agar plate and a selective medium such as a tellurite plate (e.g., cystine-tellurite blood agar or modified Tinsdale’s medium) and incubated at 37 °C in 5% CO2 .
• Non β hemolytic region on blood agar.
• Tellurite inhibits the growth of most upper respiratory tract bacteria and gram-negative rods and is reduced by C. diphtheriae, producing characteristic gray to black color on agar.
• Degradation of cysteine by C. diphtheriae cysteinase activity produces a brown halo around the colonies.
• Tinsdale medium is the best medium for recovering C. diphtheriae in clinical specimens, but it has a short shelf life and requires addition of horse serum.
• C. diphtheriae belongs to the Hazard group 2 organisms which might require Containment Level 3 conditions for processing in some cases due to aerosol production.
• Members of C. diphtheriae have been divided into four different biotypes based on their colony morphology on different agar medium: gravis, mitis, intermedius and belfanti.

Toxigenicity testing

• All isolates of C. diphtheriae should be tested for the production of exotoxin .
• The gold standard for detection of diphtheria toxin is an in-vitro immunodiffusion assay by Elek-Ouchterlony immunodiffusion test (Elek test) .
• An alternative method is detection of the exotoxin gene using a polymerase chain reaction (PCR) based nucleic acid amplification method . This test can detect the tox gene in clinical isolates and directly in clinical specimens (e.g., swabs from the diphtheritic membrane or biopsy material). A positive culture result confirms a positive PCR assay result. A negative culture result after antibiotic therapy along with a positive PCR assay result suggests that the patient probably has diphtheria. Although this test is rapid and specific, strains in which the tox gene is not expressed can give a positive signal.
• Enzyme-linked immunosorbent assays can be used to detect diphtheria toxin from clinical C. diphtheria isolates. An immunochromatographic strip assay allows detection of diphtheria toxin in a matter of hours. However, these assays are not widely used.

Antibody detection

• Measurement of antibodies to diphtheria toxin in serum collected before administration of antitoxin may support the diagnosis when cultures are negative .
• Serological tests include enzyme immunoassay, neutralization test, and passive hemagglutination assay.

• The World Health Organization (WHO) defines protective levels of antitoxin as ≥0.01 IU/mL.

  Treatment of Corynebacterium diphtheriae

The treatment of Corynebacterium diphtheriae infection involves two main aspects: neutralizing the toxin and eradicating the bacteria. The following are the main steps involved in the treatment of Corynebacterium diphtheriae infection:

• Antitoxin administration: The most important and urgent step in the treatment of Corynebacterium diphtheriae infection is the administration of diphtheria antitoxin (DAT) to neutralize the circulating toxin that is not bound to tissues. DAT is a horse-derived immunoglobulin that contains antibodies against diphtheria toxin. DAT should be given intravenously as soon as the clinical diagnosis of diphtheria is made, without waiting for laboratory confirmation. The dose of DAT depends on the severity and duration of the disease, ranging from 10,000 to 100,000 units. DAT can cause hypersensitivity reactions in some patients, so a skin test should be performed before administration. DAT can also transmit zoonotic infections, such as tetanus.
• Antibiotic therapy: Antibiotics are used to inhibit the growth of Corynebacterium diphtheriae and prevent further toxin production. Antibiotics also help to eliminate the bacteria from the throat and skin, reducing the risk of transmission to others. The antibiotics of choice are penicillin or erythromycin, which are effective against most strains of Corynebacterium diphtheriae. The recommended doses are 300,000 units of penicillin G intramuscularly every 12 hours for 14 days, or 500 mg of erythromycin orally every 6 hours for 14 days. Some strains of Corynebacterium diphtheriae may be resistant or tolerant to penicillin or erythromycin, so susceptibility testing is recommended. Other antibiotics that may be used include macrolides, rifampin, and fluoroquinolones.
• Supportive care: Patients with Corynebacterium diphtheriae infection may require supportive care to manage the complications of the disease, such as respiratory obstruction, cardiac arrhythmias, myocarditis, neuropathy, and renal failure. Supportive care may include oxygen therapy, mechanical ventilation, cardiac monitoring, fluid and electrolyte replacement, antipyretics, analgesics, and corticosteroids . Patients with Corynebacterium diphtheriae infection should be placed in strict isolation to prevent secondary spread and should be monitored daily for signs of deterioration.
• Prevention: The best way to prevent Corynebacterium diphtheriae infection is through active immunization with diphtheria toxoid, which induces protective antibodies against diphtheria toxin. Diphtheria toxoid is usually combined with tetanus toxoid and pertussis vaccine (DTaP) for infants and children, or with tetanus toxoid and reduced-dose pertussis vaccine (Tdap) for adolescents and adults. The recommended schedule for immunization is three doses in the first year of life, followed by a booster dose at 15-18 months and another booster dose at 4-6 years. A single dose of Tdap is recommended for adolescents aged 11-12 years and for adults who have not previously received it. Booster doses of Td (tetanus and diphtheria toxoids) are recommended every 10 years for adults. In addition to immunization, other preventive measures include contact tracing and prophylaxis of close contacts of patients with Corynebacterium diphtheriae infection. Contacts should receive a dose of DAT if they have not been fully immunized or if they have low levels of antitoxin antibodies. Contacts should also receive a dose of antibiotic (penicillin or erythromycin) and a booster dose of diphtheria toxoid if they have not received one in the past 5 years.

Prevention and control of Corynebacterium diphtheriae

The best way to prevent and control diphtheria is through vaccination. There are four vaccines that protect against diphtheria: DTaP, Tdap, DT, and Td. These vaccines also protect against tetanus, and DTaP and Tdap also protect against pertussis (whooping cough). The vaccines are given as a series of shots at different ages and intervals. The following are the general recommendations for diphtheria vaccination:

• Infants and children should receive five doses of DTaP vaccine at 2, 4, 6, 15-18 months, and 4-6 years of age.
• Preteens should receive a booster dose of Tdap vaccine at 11-12 years of age.
• Adults who have never received Tdap vaccine should get one dose, especially if they are in close contact with infants or pregnant women.
• Pregnant women should receive a dose of Tdap vaccine during each pregnancy, preferably between 27 and 36 weeks of gestation.
• Adults should receive a booster dose of Td vaccine every 10 years.

Vaccination not only protects the individual from getting sick, but also prevents the spread of the bacteria to others. However, some people may still get infected with C. diphtheriae despite being vaccinated, especially if they have not completed the recommended vaccine series or if their immunity has waned over time. Therefore, it is important to monitor for signs and symptoms of diphtheria and seek medical attention promptly if suspected.

In addition to vaccination, other preventive measures include :

• Isolating patients with respiratory or cutaneous diphtheria in a hospital setting until they are no longer infectious.
• Giving antibiotics to close contacts of patients with diphtheria to prevent them from getting sick or spreading the bacteria. Close contacts include household members, people who have had frequent or close contact with the patient, or people who have been exposed to the patient`s respiratory secretions or skin lesions. Antibiotics such as erythromycin, azithromycin, clarithromycin, or penicillin are effective in eliminating C. diphtheriae from the throat or skin. Antibiotics should be given for 7 to 10 days and followed by a throat swab culture to confirm clearance of the bacteria.
• Administering diphtheria antitoxin to patients with suspected or confirmed diphtheria as soon as possible. Diphtheria antitoxin is a serum that contains antibodies that neutralize the toxin produced by C. diphtheriae. It can reduce the severity of symptoms and prevent complications such as myocarditis or neuropathy. Diphtheria antitoxin is given intravenously or intramuscularly depending on the severity of the disease. The dose is determined by the extent of the pseudomembrane and the duration of illness.
• Practicing good hygiene such as washing hands frequently, covering coughs and sneezes, and avoiding sharing personal items with others.
• Reporting cases of diphtheria to public health authorities for surveillance and outbreak response.

Diphtheria is a serious and potentially fatal disease that can be prevented by vaccination and controlled by early diagnosis and treatment. By following these measures, we can reduce the risk of diphtheria infection and transmission in our communities.

The best way to prevent and control diphtheria is through vaccination. There are four vaccines that protect against diphtheria: DTaP, Tdap, DT, and Td. These vaccines also protect against tetanus, and DTaP and Tdap also protect against pertussis (whooping cough). The vaccines are given as a series of shots at different ages and intervals. The following are the general recommendations for diphtheria vaccination:

• Infants and children should receive five doses of DTaP vaccine at 2, 4, 6, 15-18 months, and 4-6 years of age.
• Preteens should receive a booster dose of Tdap vaccine at 11-12 years of age.
• Adults who have never received Tdap vaccine should get one dose, especially if they are in close contact with infants or pregnant women.
• Pregnant women should receive a dose of Tdap vaccine during each pregnancy, preferably between 27 and 36 weeks of gestation.
• Adults should receive a booster dose of Td vaccine every 10 years.

Vaccination not only protects the individual from getting sick, but also prevents the spread of the bacteria to others. However, some people may still get infected with C. diphtheriae despite being vaccinated, especially if they have not completed the recommended vaccine series or if their immunity has waned over time. Therefore, it is important to monitor for signs and symptoms of diphtheria and seek medical attention promptly if suspected.

In addition to vaccination, other preventive measures include :

• Isolating patients with respiratory or cutaneous diphtheria in a hospital setting until they are no longer infectious.
• Giving antibiotics to close contacts of patients with diphtheria to prevent them from getting sick or spreading the bacteria. Close contacts include household members, people who have had frequent or close contact with the patient, or people who have been exposed to the patient`s respiratory secretions or skin lesions. Antibiotics such as erythromycin, azithromycin, clarithromycin, or penicillin are effective in eliminating C. diphtheriae from the throat or skin. Antibiotics should be given for 7 to 10 days and followed by a throat swab culture to confirm clearance of the bacteria.
• Administering diphtheria antitoxin to patients with suspected or confirmed diphtheria as soon as possible. Diphtheria antitoxin is a serum that contains antibodies that neutralize the toxin produced by C. diphtheriae. It can reduce the severity of symptoms and prevent complications such as myocarditis or neuropathy. Diphtheria antitoxin is given intravenously or intramuscularly depending on the severity of the disease. The dose is determined by the extent of the pseudomembrane and the duration of illness.
• Practicing good hygiene such as washing hands frequently, covering coughs and sneezes, and avoiding sharing personal items with others.
• Reporting cases of diphtheria to public health authorities for surveillance and outbreak response.

Diphtheria is a serious and potentially fatal disease that can be prevented by vaccination and controlled by early diagnosis and treatment. By following these measures, we can reduce the risk of diphtheria infection and transmission in our communities.

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