Corynebacterium diphtheriae: morphology, characteristics, pathogenesis, diseases, lab diagnosis, vaccine and treatment

Corynebacterium diphtheriae

• Corynebacteria are classified as Actinobacteria and are gram positive in nature.
• They are rod shaped bacteria that survives in aerobic environment and are non-motile.
• Phylogenetically, Corynebacteria are related to mycobacteria and actinomycetes.
• A diverse community of bacteria, including animal and plant pathogens, as well as saprophytes, make up the genus Corynebacterium.
• Some of the corynebacteria are part of the normal human flora, particularly the skin and nares, that tends to find an appropriate niche in almost every anatomical location.
• Corynebacterium diphtheriae, the causal agent of the disease diphtheria, is the best known and most widely studied species.
• The diphtheria bacillus is also termed as Klebs-Loffler bacillus as it was first observed and explained by Klebs in 1883 and was first cultivated by Loffler in 1884.
• The pathogenecity of C. diphtheriae is due to the development of a potent exotoxin active in a number of tissues, including the muscles of the heart and peripheral nerves.

Morphology of the Corynebacterium diphtheriae:

• They are thin, slender gram-positive bacilli, but, especially in old cultures, they are easily decolorized.
• They measure about 3-6 μm × 0.6-0.8 μm.
• They have a remarkable ability to clubbing at one or both ends.
• They are largely pleomorphic.
• Cells often exhibit septa, and branching is seldom observed.
• They are nonmotile, nonspore forming, and nonacid fast.
• The bacilli are arranged in smears in a characteristic fashion.
• They are typically seen in pairs, palisades (resembling stakes of a fence) or small groups or as individual cells lying at sharp angles to another, resembling the letters V or L.
• This unique arrangement with C. diphtheriae has been termed the Chinese letter or cuneiform arrangement.
• This is because of the incomplete separation of the daughter cells after binary fission.
• This organism has granular and irregular staining.
• The granules in the cell are metachromatically reddish-purple when stained with methylene blue or toluidine blue.
• These granules are termed as metachromatic granules, volutin granules or Babes-Ernst granules.
• They are often located at the poles of the bacilli and are termed as polar bodies.
• To clearly show the granules, unique stains such as Albert’s Neisser’s and Ponder’s have been devised.
• The granules are stained bluish black and the protoplasm green with Albert’s stain.
• The granules indicate the accumulation of polymerized polyphosphates.
• The development of granules is best seen on the Loffler’s serum slope.
•  They seem to represent storage depots for materials required to form high-energy phosphate bonds.
• Three strains of Corynebacterium diphtheriae are known, gravis, intermedius and mitis.
• They are listed here as the severity of the disease they produce in humans in decreasing order.
• The same toxin is produced by all strains and is capable of colonizing the throat.
• The variations in virulence between the three strains can be understood by their diverse abilities to produce the toxin in rate and quantity, and by their varying growth rates.
• The gravis strain has 60 minutes of generation time (in vitro); the intermedius strain has about 100 minutes of generation time; and the mitis stain has about 180 minutes of generation time.
• Usually, the faster growing strains generate a larger colony on most growth media.
• A faster growth rate in the throat (in vivo) may enable the organism to deplete the local supply of iron more rapidly in the invaded tissues, allowing for earlier or greater development of diphtheria toxin.
• Also if bacterial growth kinetics are accompanied by the kinetics of toxin development, the faster growing variety will reach an efficient toxin level before the slow growing varieties.

Cultural Characteristics of Corynebacterium diphtheriae :

• C. diphtheriae is an aerobe and facultative anaerobe.
•  The optimum temperature for growth is 37°C and optimum pH is 7.2.
• For primary isolation and characterization, complex media are required.
• Growth can occur on ordinary nutrient agar, however, its growth is enhanced by the presence of animal proteins like blood or serum.
• Two media are essential for this purpose:
  • Löffler’s serum slope.
  • Blood agar having  fresh, lysed or heated blood.

Löffler’s Serum Slope :

• On Löffler’s serum slope, diphtheria bacilli develop very quickly and colonies can be seen in 6-8 hours, long before other bacteria grow.
• Colonies are tiny circular white opaque disks at first but on continuous incubation they expand and may develop a distinct yellow tint.
• The medium of Löffler is also helpful because it does not support the growth of streptococci and pneumococci that may be present in the clinical sample and limit the activity of most oral commensals and retards the growth of others, behaving as a selective agent but having little effect on diphtheria bacilli, such as Candida albicans and Staphylococcus aureus.

Tellurite Blood Agar

• The introduction of potassium tellurite(0.03-0.04%) helps to make the medium selective for corynebacteria by suppressing most other pathogenic and commensal bacteria.
• C. diphtheriae exhibit gray/black, shiny or dull black colonies.
• This is because the tellurite ion passes through the cell wall and membrane into the cytoplasm, and here, it is reduced to the metal tellurium and is precipitated inside the cells.
• The tellurite salts are reduced by any other corynebacterial, yeasts and staphylococci producing distinctive gray to black colonies.
• The addition of cystine to a medium containing tellurite (Tinsdale’s medium) has significantly aided to isolate diphtheria bacilli.
• The growth of diphtheria bacilli on the tellurite medium may be delayed and it may take two days for colonies to appear.

Biochemical test for Corynebacterium diphtheriae

• C. diphtheriae degrades glucose and maltose along with the production of acid (but no gas).
• However, it cannot ferment mannitol, lactose, trehalose or sucrose.
• C. diphtheriae is H₂S positive and reduces nitrate to nitrite.
• It does not liquefy gelatin nor hydrolyze urea.
•  It does not form phosphatase as well.

Pyrazinamidase (PYZ) Test:

• In pyrazinamidase (PYZ) test, the organisms that are capable of producing pyrazinamidase (PYZ) converts pyrazinamide into pyrazinoic acid.
• This test is esssential to differentiate C. diphtheriae (PYZ-negative) from other corynebacterium species (mostly PYZ-positive).
• Exception are C. ulcerans and C. pseudotuberculosis which are also PYZ-negative but they are urease test positive which differentiate them from C. diphtheriae (urease negative).

Diphtheria Toxin

• A very powerful exotoxin is produced by toxigenic strains of C. diphtheriae
• The toxicity found in diphtheria is specifically related to the toxin secreted by the bacteria at the site of infection.

Lysogeny and Toxin Production:

• Corynephages (tox⁺) acts as the genetic determinant that controls the toxin production.
• The toxigenicity of the diptheria bacillus relies on the presence of corynephages (tox⁺).
• By infection with the related bacteriophage, non-toxigenic strains can be transformed to tox+.
• This is called as lysogenic conversion or phage conversion.          
• The bacillus loses its toxigenicity when the bacillus is cured of its phage, as by developing it in the presence of antiphage serum.                                                                                                                                           

Iron for Toxin Production

• The production of toxins is also affected by the iron concentration in the medium.
• For toxin production, the optimal level of iron is 0.1 mg per liter, while a concentration of 0.5 mg per liter inhibits toxin formation.
• The toxin is released in massive quantities only when the obtainable iron in the culture medium is depleted.

Properties of Diphtheria Toxin:

• Diphtheria toxin is a crystalline, heat-labile protein that is iron-free.
• It is highly potent and is fatal for humans in amounts of 130 ng per kg of body weight.
• The lethal dose of diphtheria toxin is 0.1 μg/kg or less in highly susceptible animals (guinea pig, rabbit).
• The diphtheria toxin has a molecular weight of around 62,000 and is a protein.
• Fragment A seems to have all the enzymatic activity where-as fragment B is accountable for binding the toxin to the cells.
• This toxin is thermo-labile.
• It is highly potent.
• In eukaryotic cells, the toxicity of the toxin is due to its capacity to inhibit protein synthesis.
• Two fragments A (active) and B (binding) of MW 24,000 and 38,000, respectively, constitute the toxin.
• For the toxic impact, both fragments are required.

Mode of Action of Diphtheria toxin :

• The bacterial cell secretes the toxin and is non-toxic unless exposed to trypsin.
• Two polypeptide fragments, A and B, which are connected together by a disulfide bridge, result from trypsinization.
• Fragment A is responsible for cytotoxicity; fragment B binds to eukaryotic cell receptors and mediates the cytoplasm entry of fragment A.
• By resisting the binding of the toxin to the cells, the antibody to fragment B is defensive.
• By inhibiting protein synthesis, the diphtheria toxin works.
• Primarily, fragment A separates nicotinamide adenosine dinucleotide (NAD) to form nicotinamide and adenosine diphosphoribose (ADPR).  
• ADPR binds to and deactivate elongation factor 2 (EF-2), an enzyme that is vital for the elongation of ribosome polypeptide chains.
• Protein synthesis inhibition is possibly responsible for both the toxin’s necrotic and neurotoxic impact.
• We may summarize the reaction as follows:
• NAD+ + EF-2 (active) = ADPR-EF-2 (inactive) + nicotinamide + H+

Resistance in Corynebacterium diphtheriae:

• C. Diphtheriae is more resistant than most non-spore forming bacilli to light, desiccation, and freezing action.
• Organisms live for at least 14 weeks on dried fragments of pseudomembranes.
• Nevertheless, they are killed by a 1-minute exposure to 100 ° C or a 10-minute exposure to 58^oC.
• They are sensitive to most of the regularly used disinfectants.
• In 0.85 percent NaCl solution, it dies rapidly, but stays alive in dust and fomites for weeks when dry and shielded from sunlight.
• It is prone to penicillin, erythromycin and broad spectrum antibiotics.

Antigenic Structure of Corynebacterium diphtheriae :

• Antigenically, diphtheria bacilli are heterogeneous.
• They carry three distinct antigens:
• A deep-seated antigen found in all corynebacterial species as well as in Mycobacterium tuberculosis
• A heat-labile protein (K antigen).
• A heat-stable polysaccharide (O antigen).

Pathogenesis of Corynebacterium diphtheriae:

• Diphtheria bacilli creates an inflammatory exudate in the upper respiratory tract and causes necrosis of the faucial mucosal cells.
• By destroying epithelial cells or neutrophils, the diphtheria toxin may help colonization of the throat or skin.
• Diphtheria is a toxemia.
• Organisms do not penetrate deep into the mucosal tissue and there is generally no bacteremia.
• Exotoxin is locally produced and distributed to distant organs via the bloodstream, with a particular affinity for the heart muscle, the peripheral nervous system, and the adrenal glands.
• The pathogenesis process can be summarized in two main steps:
• Invasion:
  • Colonization and subsequent bacterial proliferation leads to the invasion of the local tissues.
  • On the adherence mechanisms of C. diphtheriae, little is understood, however, many forms of pili are produced by it.
  • The toxin of diphtheria, as well may be involved in throat colonization.
• Toxigenesis:
  • The development of a toxin by bacteria is termed as toxigenesis.
  • By inhibiting protein synthesis in the cells, the diphtheria toxin triggers the death of eucaryotic cells and tissues.
  • Although the toxin is responsible for the lethal symptoms of the disease, toxigenicity alone cannot be correlated with the virulence of C diphtheriae, because toxigenesis is apparently followed by a distinct invasive process.
  • Due to short-range effects at the colonization site, the diphtheria toxin plays an important role in the colonization process.

Diseases caused by Corynebacterium diphtheriae :

• The main disease caused by C. diphtheriae is diphtheria. The Greek meaning of diphtheria refers to the leathery skin referring to the pseudo-membrane that is formed on the pharynx initially.
• Occasionally, this microorganism is responsible for wound and chronic skin infections.
• Throughout the world, the non-toxicogenic strains are linked with the endocarditis, cerebral abscess, meningitis and osteoarthritis.

i. Diphtheria:

• As described by CDC, diphtheria is a disease of the upper respiratory tract marked by sore throat, low fever, and an adherent membrane (called a pseudo-membrane on the tonsils, pharynx, and/or nasal cavity).
• Myocarditis, polyneuritis, and other systemic toxic effects may be caused by diphtheria toxin developed by C. diphtheriae.
• A relatively milder form of diphtheria can be limited to the skin.
• Diphtheria is an infectious disease spread either by direct physical contact or inhaling the aerosolized secretions of infected individuals.
• Diphtheria which was once much prevalent has largely been eliminated in developed countries by the use of the DPT vaccine.
•  Diphtheria is a quickly evolving, acute febrile infection which includes both local and systemic pathology.
• In the upper respiratory tract, a local lesion occurs and involves necrotic epithelial cell damage.
• Blood plasma spills into the area as a result of this injury and forms a fibrin network that is interlaced with fast-growing C. diphtheriae cells.
• This membranous network, referred to as a pseudo-membrane, covers the site of the local lesion, causing respiratory distress, including suffocation.
• The diphtheria bacilli do not appear to conquer tissues below or away from the surface epithelial cells at the zone of the local lesion.
• Though at this site they generate the toxin that is absorbed and circulated through lymph channels and blood to the prone tissues of the body.
• The systemic pathology of the disease results from degenerative alterations in these tissues, which consists of the heart, muscle, peripheral nerves, adrenals, kidneys, liver and spleen.

ii. Systemic Effects:

• The toxin is also absorbed and may cause a number of systemic effects affecting the kidneys, heart, and nervous system, as all tissues have the receptor of toxin and will be affected.
• Intoxication takes the form of peripheral neuritis and myocarditis and can be related to thrombocytopenia.
• There is also visual disturbance, trouble swallowing and paralysis of the arms and legs, but it usually recovers spontaneously.
• Myocarditis can result in complete heart block.
• Death is mainly caused by congestive heart failure and cardiac arrhythmias.

iii. Complications:

• The prominent complications are:
• The pseudomembrane causes the mechanical obstruction of the respiratory passage that results in asphyxia for which an emergency tracheostomy might be essential.
• Acute circulatory failure, which can be either cardiac or peripheral.
• Postdiphtheritic paralysis, that usually occurs in the third or fourth week of the disease; palatine and ciliary but not pupillary paralysis is indicative, and spontaneous recovery is the rule.
• Septic conditions such as pneumonia and otitis media arises.

iv. Cutaneous Diphtheria:

• In case of the cutaneous diphtheria that is present in the tropics, the toxin is also absorbed systematically but systemic complications are less likely as compared to the upper respiratory infections with C. diphtheriae.

Laboratory Diagnosis of Corynebacterium diphtheriae:

• The clinical experience is supported by diagnostic laboratory tests and is of epidemiological importance, but not for the treatment of specific cases.
• Without waiting for laboratory testing, specific care should be instituted immediately on suspicion of diphtheria.
• Any postponement can be fatal.
• Laboratory diagnosis consists of diphtheria bacillus isolation and evidence of its toxicity.

1. Specimens

• Before antimicrobial drugs are given, swabs must be taken from the nose, throat or other suspected lesions.
• In suspected cases of facial or nasal diphtheria, swabs should be taken from both the throat and the nose and, usually, two swabs should be taken from the most affected area.
• Swabs should also be taken from skin lesions and wounds where there is a possible diphtheria infection, and swabs should be taken from suspected carriers from both the throat and nose.

2. Microscopy:

• As C. diphtheriae is morphologically similar to other coryneforms, direct microscopy of a smear is inaccurate.
• Smears stained with alkaline methylene blue or Gram stain exhibit beaded rods in typical arrangement.
• Smear examination alone is therefore not sufficient for the diagnosis of diphtheria, but it is essential for the identification of Vincent’s angina.
• For Vincent’s spirochetes and fusiform bacilli, a Gram or Leishman stained smear is studied.
• Toxigenic diphtheria bacilli may be detected in smears by immunofluorescence.

3. Culture

• On the Löffler’s serum slope, tellurite blood agar, and blood agar, the swab should be inoculated.
• At 37^oC, the cultures should be incubated aerobically.
• It should be kept moistened with sterile horse serum unless the swab can be inoculated promptly, so that the bacilli can remain viable.
• i. Löffler’s Serum Slope
  • A growth smear from all parts of the slope mixed in the condensation water is made and after incubation for 6 hours or overnight, it is stained with the Albert-Laybourn method and  the presence of slender green-stained bacilli containing the purple-black granules characteristic of C. diphtheriae is looked.
  • The Löffler slant can yield organisms with typical “diphtheria-like” morphology in 12 to 18 hours.
• ii. Tellurite Blood Agar
  • Since growth may sometimes be delayed, blood tellurite agar is examined after 24 hours and after 48 hours.
• iii. Blood Agar
  • It is used for distinguishing streptococcal or staphylococcal pharyngitis, that may simulate diphtheria.

4. Identification Tests:

• Identification is based on fermentation reactions of carbohydrates and enzymatic activities.
• diphtheriae ferments glucose and maltose, producing acid but not gas, and is catalase positive.
• Nitrate is reduced to nitrite and is non-motile.
• Reliable identification is provided by commercial kits such as the API Coryne strip.

5. Virulence Tests:

• Before the bacteriologic diagnosis of diphtheria is definite, any diphtheria-like organism cultured must be sub-mitted to a’ virulence’ test.
• These tests are actual toxigenicity tests for an isolated diphtheria-like organism.
• Diphtheria diagnosis depends on demonstrating that diphtheria toxin is produced by the isolate.
• In vivo or in vitro methods may be used for virulence testing.
• It is rare to perform in vivo testing because the in vitro methods are reliable, less costly and free of the need to use animals.
• A. In Vivo Tests
  • Subcutaneous test.
  • Intracutaneous test.
• B. In Vitro Test
  • Precipitation test.
  • Tissue culture test.
  • Enzyme-linked immunosorbent assays.
  • Polymerase chain reaction (PCR).

Epidemiology of Corynebacterium diphtheriae:

• Diphtheria is a disease that is widely distributed, particularly in poor urban areas where crowding occurs and vaccine-induced immunity has a low protective level.
• C. diphtheriae is preserved in the population as the asymptomatic carriage occurs in the oropharynx or on the skin of immune people.
• The infection is limited to humans and usually requires contact with a case of diphtheria or a carrier.
• Humans are the only recognized reservoir, with oropharynx or skin surface carriage.
• Diphtheria is predominantly a pediatric disorder, but in areas where there are successful immunization services for infants, the highest prevalence has moved towards older age groups.
• Acquired diphtheria immunity is mainly due to the toxin-neutralizing antibody (antitoxin).
• Passive immunity in utero is gained transplacentally and can last for 1 or 2 years after birth.
• Skin infection with toxigenic C. diphtheriae (cutaneous diphtheria) also occurs.

Prophylaxis for Diphtheria:

• Active, passive or mixed are the immunization methods available.
• Of these only active immunization will provide herd immunity and result to elimination of the disease.
• Passive and combined immunization can provide emergency protection only to susceptible risk-exposed individuals.

Active Immunization for Corynebacterium diphtheriae:

• For active immunization , following preparations are used:
  • Toxin-antitoxin mixture: It is not without hazards.
  • Single vaccines: It is less frequently used.
  • Combined preparations.
• Combined Preparations:
  • DPT (diphtheria­pertussis­tetanus) vaccine  
  • DT (diphtheria­tetanus, adult type)• DT (diphtheria­tetanus toxoid)
• DPT Vaccine
  • Diphtheria toxoid is typically administered as a tri-valent preparation containing tetanus toxoid in children and pertussis vaccine is also known as the DTP, DPT or triple vaccine.
  • In immunization systems, the WHO advises that only adjuvant DPT preparations be used.
  •  Primary immunization schedule: The primary immunization schedule for babies and children consists of DPT administered at 6 weeks, 10 weeks, 14 weeks and 16/24 months of age, accompanied by a booster dose of DT at 5-6 years of age.
  • Reactions: Fever and mild local reactions following DPT immunization are prevalent.
  • Neurological (encephalitis/encephalopathy, excessive convulsions, infantile spasms and Reye’s syndrome) are the most serious complications following DPT immunization.
• Passive Immunization
  • This is an emergency method to be used, such as when a case of diphtheria is admitted to general pediatric wards, where vulnerable (non-immunized) are exposed to infection.
  • It consists of 500-1000 units of antitoxin (antidiphtheritic serum,) subcutaneous administration.
  • Precautions against hypersensitivity should be observed, as this is a horse serum.
• Combined Immunization
  • This consists of the administration of the first dose of the adsorbed toxoid, while the ADS is administered to the other arm, such that the complete course of active immunization continues, provided that the protection granted by passive immunization is short-lived.
  • Ideally, combined immunization should be provided to all cases that receive ADS prophylactically.

Treatment for Corynebacterium diphtheriae:

• Specific diphtheria treatment consists of antitoxic therapy and antibiotic therapy.
• As soon as clinical diagnosis is made, antitoxin should be given immediately to neutralize the produced toxin, since antitoxin is ineffective if given after the toxin is bound to cell receptor sites.
• The dosage suggested is 20,000 units intramuscularly for moderate cases and 50,000 to 100,000 units for severe cases, half the dose being given intravenously.
• Diphtheriae is susceptible to most antibiotics, including penicillin and erythromycin, and is used in both carriers and patients.
• The circulating toxin is not neutralized by antibiotics.
• By killing diphtheria bacilli, they stop further toxin production.
• Erythromycin can be given to penicillin-sensitive individuals.
• In the treatment of carriers, Erythromycin is more active than penicillin.