CORYNEBACTERIUM
DIPHTHERIAE
Introduction
In this case study, we initially were presented with a case where a 2-year-old male child who had not had any immunisations. He experienced an upper respiratory tract infection experiencing anorexia and lethargy. This progressed into having a high temperature at 39.9 and physical symptoms including exudative pharyngitis and enlarged lymph nodes. He was given penicillin which did not improve the situation. His condition continued to decline with increase lethargy and respiratory distress, also presenting with a high temperature of 38.9 and had exudate on the pharynx what was of a yellowish thick membrane. This was
scraped, removed and analysed through gram staining as well as being grown on cystine- tellurite blood agar.
History
Diptheria was discovered in 1884 by two German bacteriologists Klebs and Loeffler hence the bacteria is also known as Klebs-Loeffler bacillus. Loeffler was the first to find in 1884 that the diphtheriae bacteria can only be cultured and grown when retrieved from the nasopharyngeal cavity (Murphy, 2020). Loeffler found that the bacteria caused damage to the internal organs using a soluble toxin that the bacteria created while causing the disease (Murphy, 2020).
Figure 1: Preparation diphtheria anti-toxin from the white blood cells of horses in Marburg Germany in 1895 (Diphtheria, 2020).
In 1888 experiments were done on animals where they were given a sterile form of C. Diphtheriae, the showed that the pathology of the organs in the animals was identical to that of humans. This experiment showed that the exotoxin produced was the main virulence factor of the disease (Hien and White, 2014). It was in 1890 that a form of anti-toxin was produced from horses it was produced by injecting the horse with high amounts of diphtheriae toxin which then caused an immune response in the horse and white blood cells could be processed to make an anti-toxin that helps to stop the progression of the disease (Diphtheria, 2020).
The anti-toxin could not prevent the disease or stop it from spreading. Bela Schick developed a test called the Schick test in 1910 which could be used to detect if the person has immunity towards Diptheria it was used to give out vaccinations to people who did not have immunity. During the start of the 19th century in the early 1900s, many attempts of making vaccines or toxoid for diphtheria was made using toxin-antitoxin mixtures, it was only in 1921 after approximately 22 years that a toxoid was discovered (Millward, 2020). Even though a toxoid was discovered in 1921, its widespread use was only established in the early 1930s, and from the year 1940, it was incorporated into the pertussis vaccine and tetanus toxoid and used more regularly (Millward, 2020).
Children below the age of 15 were the ones that were affected heavily by the spread of diphtheria. The spread of diphtheria was very prominent in countries such as England and Wales wherein these countries there were years where more than 40,000 children were killed by diphtheria. After the widespread use of the toxoid the rate of death had decreased from around 40,000 to 962, it was the first free vaccine that was administered to the British public (2020). In 1974 the WHO (World Health Organisation) decided to add vaccination for diphtheria into their extended immunisation program for developing countries (Timeline | History of Vaccines, 2020).
During 1949 due to improper manufacturing of vaccine aluminium phosphate toxoid many children died. It took more than 50 years for effective immunisation of diptheria to occur as the widespread use of the toxoid started around 1949 and effective immunisation was achieved in the early 2000s (Timeline | History of Vaccines, 2020).
One of the largest outbreaks that had occurred recently is the diphtheria outbreak in the Soviet Union which was from 1990-98 there were more than 155,000 cases and around 5,000 deaths that had occurred during the eight years of the large outbreak in the Soviet Union that has been last recorded incidence of a large outbreak, in the present-day few cases are recorded mostly a rare occurrence in developed countries (Murphy, 2020).
Infectious Agent
Corynebacterium diphtheriae is a non-motile, non-encapsulated, non-sporulating gram-positive rod-shaped bacterium with a high Guanine-cytosine content (Konig, C et al, 2014). C. diphtheriae is genetically heterogeneous and has four biovars which are defined as Gravis, Mitis, Belfanti and Intermedius (Thompson et al, 1983).
As shown below in table 1, C. diphtheriae had a GC content of 53% and a genome size of 2,488,635. In addition, this table shows that C. effeiciens and C. glutamicum have higher GC content, a larger genome size in comparison to C. diphtheriae and they also produce glutamate. C. diphtheriae does not produce glutamate due to the fact that it does not come from glutamic acid-producing species.
However, in the C. diphtheriae genome, the high Guanine-cytosine content is not constant throughout the whole genome as there have been findings that show that in the region around the terminus there is a significantly low high Guanine-cytosine content, and this reflects on its genetic diversity (Cerdeno-Tarraga, 2003). This is the cause of the 102 strains of C. diphtheriae and change in genome (Titov et al , 2003).
Diphtheriae toxins have 3 structural domains and each of them have a different biological function implicated in the intoxication of cells. The functions vary from cell surface binding, internalisation into endosomes and then crossing the endosome membrane to go into the cytosol and blocking cellular protein synthesis (Gillet et al, 2015). Moreover , diphtheriae toxins have a crystal structure which consists of 1,046 amino acid residues , including 2 bound adenylyl 3′‐5′ uridine 3′ monophosphate molecules and 396 water molecules (Bennett and Eisenberg, 1994).
In the cell wall of C. diphtheriae, it contains an arabinogalactan polymer that anchors an outer lipid‐rich domain to the murein sacculus of the cell. Alpha-alkyl, beta-hydroxy fatty acids and corynomycolic acids are produced through a Claisen-like condensation reaction between two fatty acyl chains. Moreover, there are pilli found on the exterior and sortase-like proteins are produced to help with anchoring of the pilli and polymerising them (Cerdeno-Tarraga, 2003) . As shown in figure 1, SpaA-type pilus is composed of a major pilin subunit SpaA and this is important for the formation of the pilus structure. pilin motif and the sorting signal are both necessary and sufficient to promote pilus polymerization by a process requiring the function of a pilus-specific sortase (Mandlik et al , 2010).
As shown in figure 1, DIP0733 is a transmembrane protein. DIP0733 is a multi-functional virulence factor of C. diphtheriae. It has also been found that DIP0733 may play a role in avoiding recognition of c. diphtheriae by the immune system and this protein is involved in adhesion, invasion of epithelial cells and induction of apoptosis. Moreover, DIP0733 has a c-terminal coiled coil domain which is essential when it bind to type I collagen, fibronectin and human plasma fibrinogen. Another protein known as DIP2093 is a SDR protein and is also involved in binding to Type I collagen and host colonisation (Antunes et al, 2015).
Moreover, CdiLAM is an adhesin of C. diphtheriae in the first step of infection to human respiratory epithelial cells. The key structural features of CdiLAM are the linear α-1-6-mannan with side chains containing 2-linked α-D-Manp and 4-linked α-D-Arafresidues (Moreira et al, 2008).
Pathogenicity
Diphtheria toxin is a protein produced by pathogenic Corynebacterium diphtheriae strains.
Toxigenic strains lead to the disease by secreting and increasing number of diphtheria exotoxin in either nasopharyngeal or skin lesions.
This can spread from person to person via respiratory droplets such as from sneezing or coughing (Murphy, 2020).
This exotoxin produced by Corynebacterium deactivates eukaryotic translation elongation factor 2 by binding to it, which then stops the translation in the protein synthesis, as a result, it leads to the eukaryotic cells to die. Furthermore, the waste products and proteins produced by toxin can cause pseudomembrane to attach with nasal tissues, larynx, pharynx, and tonsils which can block breathing.
In addition, if exotoxin increases and starts to spread in the bloodstream then it can damage remote organs, for example, kidneys, heart, and liver which can also lead to death (Wenzel et al., 2020).
This exotoxin is ‘Y’ shaped, it consists of a single polypeptide chain with 535 amino acids and it has A-B subunits combined by disulfide bridge.
Subunit A-B has 3 structural domains, where each has different biological functions.
The C domain makes up the A subunit, T, and R domains make up the B subunit.
Subunit B consists of:
1. Receptor binding domain, that has the function to recognize specific receptor found on the surface of host cells. This process allows the endocytosis of the toxin.
2. Translocation/transmembrane domain, that enters the endosome when the environment has low pH, and its function is to help transport the catalytic domain into the cytoplasm.
Subunit A consists of:
Catalytic domain, it carries toxic and functions to transport ADP-ribose from cytosolic NAD to the substrate, called elongation factor 2. This process stops cellular proteins from synthesizing as a result it leads to cell apoptosis.
Glutamic acid 148 in the active site of the C domain forms a stable bond with the nicotinamide group which allows ADP ribose to bond and attack diphthamide in EF2 (Leone, 2020).
Exotoxin is coded by tox gene, and it enters by a lysogenic bacteriophage B-phage.
The binding receptor in subunit B binds with the heparin binding epidermal growth factor receptor found on the surface of the host cells such as nerve cell or heart cells. The attachment of the receptor domain to heparin-binding epidermal growth factor and cell membrane proteins CD-9 allows the toxin to enter the cell.
A-B toxin enter the cell by endocytosis, the cell engulfs the toxin in an endosome. This causes the content of vacuole to become acidic leading A-B units to separate.
Unit A enters the cytoplasm of the cell and applies toxic effects, while unit B leaves the cell by exocytosis.
Subunit A transfer's adenine ribose phosphate from NAD to EF-2 known as ADP ribosylation. This disables EF-2 and stops protein synthesis in cells.
Lysogenised toxin synthesis is managed by a chromosomally encode osmolarity d element, diphtheria toxin repressor (DTxR) which is activated when iron concentrations increase, and it can attach to the operator of toxin gene and inhibit toxin formation. Therefore, the virulence of Corynebacterium diphtheriae disease is associated with diphtheria toxin repressor (DtxR) (Diphtheria toxin: The nuts and bolts, 2019).
Epidemiology
The global prevalence of diphtheria has reduced dramatically due to comprehensive vaccine coverage; nonetheless, the disease remains widespread in many countries, although there are few reliable estimates of prevalence in these countries (Basak et. al 2015).
A noteworthy feature of mass immunisation of diphtheria toxoid is that as the proportion of the population of protective levels of anti-toxin immunity (≥ 0.01 IU /ml) increases, the degree of exclusion of toxigenic strains from the population decreases (Saikia et. al 2010).
Until widespread immunisation of the U.S. population with diphtheria toxoid, diphtheria was usually children's disease. The introduction of the DTP vaccine programme has minimised childhood diphtheria in many countries. For example, during the first 13 years of the mass vaccination period (1919–1931), the confirmed cases of diphtheria decreased by 82.4% in the Netherlands.
A major public health impact is triggered by timely vaccination. Studies showing that 72 % of children were given their first dose (at 2 months of age) of DTP in time, however only 59% (at 6 months of age) had received the third dose (DTP-3). Timely receipt, however, of scheduled vaccine dosages for urban Australian Indigenous children (Lodeiro et. al 2016).
Local diphtheria outbreaks are almost always linked to an immune carrier who has returned from an endemic area. In reality, recent outbreaks of clinical diphtheria have been associated with visitors from Russia and Eastern Europe in America and Europe (Lodeiro et. al 2016).
The WHO registered 16,611 cases in 2018 (Fig. 1). Diphtheria is commonly underreported in many regions like the countries of Asia, Africa and the eastern Mediterranean. In a number of countries, including Nigeria in 2011 and India during 2010–2016, outbreaks of respiratory diphtheria have been recorded. Highly vulnerable, as a result of the lack of availability of public-sector health facilities, are refugee resettlement centres (e.g. in the Bangladeshi Rohingy refugee population). Estimated that approximately 76 % of refugees did not have long-term diphtheria security. Difference in Haiti, Venezuela and Yemen occurred during 2015-2018 due to the socio-economic crise or war which led to poor health and immunisation access. In endemic countries, the status of carriers of diphtheria is critical. A research conducted in India in 1989 on a reported case of C-carrying diphtheria. In~20% of children tested and 65% of species toxic is diphtheria.
However, diphtheria carriers' status is much lower, even among people who have a poor immunisation or who drink alcohol, in non-endemic countries with optimal vaccine coverage (Page et al 2019).
With a median of three cases per year, the overall incidence of diphtherias in the UK is minimal, demonstrating the effectiveness of the vaccine programme. A significant change in the proportion of dermal diphtheria, especially due to C. diphtheriae, has been a major increase, which caused this species to be a slight predominant cause agent in comparison to previous UK cases, which recorded greater numbers of C, from 1986 to 2008. diphtheriae Cases of ulcer, as recorded in other countries in Western Europe during the same time (Sharma et al. 2007).
Clinical Aspects
Laboratory tests can confirm the presence of the toxigenic C. diphtheriae in the throat, nose and skin (Torres et al., 2013). Dacron swabs are used to collect specimens from the pharyngeal tonsils, whereas calcium alginate swabs are inserted through the nares to collect nasopharyngeal swabs (Funke et al. 2012). As mentioned in ‘Pathogenicity’ toxigenic strains increase the number of diphtheria exotoxin in skin lesions. These lesions are often covered with a pseudomembrane and may have to be exposed before swabbing (Natividad et al., 2015). The specimens are then placed into a semisolid transport medium such as Tinsdale tellurite agar for primary isolation and are incubated at 37oC in 5% CO2. This is the best form of isolation as the medium has a short shelf life (Dias et al., 2011), and therefore the nutrients needed to survive would be at optimal quality.
The tellurite bacterium inhibits the growth of other upper respiratory tract bacteria (Schubert JH, et al., 2010) and can indicate whether C. diphtheriae is completely non-haemolytic. The diphtheria bacilli reduces the tellurite to metallic tellurium, producing grey or black colonies on the agar and further degradation of cysteine by the bacterium, produces a brown halo (Burkhardt, 2015), as shown in Figure 7.
As mentioned in, ‘Infectious agents’, C.diphtheriae can be characterized into different groups based on their carbohydrate fermentation patterns, such as mitis, intermedius, or gravis, as shown in Figure 8. They all produce an immunologically identical toxin. The variations in virulence between the strains are due to the rate they produce the toxin. Mitis, intermedius and gravis have a generation time (in vitro) of 180,100 and 60 minutes respectively (Efstratiou A, et.al, 2014). A larger colony is typically observed with the gravis strain, and a faster growth rate can lead to high usage of iron supply in the tissues by the bacterium, paving way for greater production of the diphtheria toxin. Therefore, categorizing the bacterium, allows the us to see the severity of the bacterium species. Mitis, intermedius and gravis are correspondingly responsible for the mild, intermediate and acute form of the disease
To assess the toxicity of these strains, the Elek culture precipitation test is routinely used to test exotoxin production. It is a vital test for diagnosis and should be carried out immediately on any suspected isolate (Burkhardt, 2015).. A filter paper strip is covered with diphtheria antitoxin and placed underneath the agar, whilst it is still liquid. Strains that are to be tested, are streaked on the surface of the agar in a line and perpendicular to the anti-toxin paper strip. After incubation at 37oC for 24 hours, the presence of fine lines at a 45-degree angle to the streaks indicates that toxins from the strains were produced and these reacted with the anti-toxin (J.Clin et.al, 2013).
As an alternative approach for detecting the exotoxin gene, PCR can also be used. 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 is likely to have diphtheria. Although this test is rapid and precise, a positive signal results may be provided in strains in which the toxic gene is not expressed (Spier SJ et.al, 2011).
To observe under a microscope, C. diphtheriae is placed on a glass side and the smears are covered in Albert’s stain for roughly 7 minutes, followed by Albert’s iodine for 2 minutes. After rinsing the glass side, it is placed under a microscope to be observed under oil immersion lens, as shown in Figure 9(C. diphtheriae and Diphtheria, 2020)
Therapies
The main way of controlling and treating C. Diphtheriae infection is with the vaccine, which is currently given in the UK at a young age. This being the DTP containing tetanus and pertussis as well as diphtheria vaccines in a single dose. Starts as early as 6 weeks of age as a series of 3 doses/injections at a minimum of 4 weeks apart, and last dose should be aimed to be given around 6 months of age. With boosters at 12-23 months, 4-7 years and 9-15 years. This can be completed up until 7 years of age. The vaccine can be given also be given later on in life (over 7 years old) just at modified intervals between doses (World Health Organization, 2018), but this level of vaccination is lacking in developing countries.
Although antibiotics and the antitoxin can be used on the onset of the symptoms, it can only be given before the toxin has enter the cells of the person infected (Baron et al., 1996). As once the infection has reached this stage, the antitoxin is unable to reach the toxin. As it cannot enter the cell and can’t stop the toxins actions.
The vaccine is made up of a diphtheria toxoid this is made from the C. Diphtheriae toxin as illustrated in Figure 11. By producing large amounts of the toxin incubating it with formatting to form the toxoid and purifying the mixture to obtain the necessary dosage. This process has been continued to be refined as more has been learnt about the diseases and the way the immune system reacts to the vaccine, by using recombinant granulocyte macrophages with the diphtheria vaccine. Which was found to improve the humoral and cellular immune response. (Grasse et al., 2018).
The effectiveness of the different antibiotics is shown in the figure 11 below. Erythromycin is a macrolide antibiotic. That works by entering the bacterial cell wall, and membrane and irreversibly binds to the 50S ribosomal subunit. Therefore, preventing protein synthesis (Usary and Champney, 2001). Clindamycin works in the same way as erythromycin, but reversibly binds to the subunit (Nodzo et al., 2019). The two penicillin-based antibiotics benzyl penicillin and amikacin as well as the antibiotic cephaloridine all effect the formation of the cell wall by binding to the Penicillin binding proteins (PBS) (Yocum et al., 1980, Acar et al., 1975)
C. Diphtheriae is showing some resistance to erythromycin this is shown in a study in 2019 in Indonesia where 2.8% of patients with diphtheria were resistant to erythromycin (Husada et al., 2019). It has been shown this is due to a mutation on the pNG2 plasmid (Tauch et al., 2003).
The antitoxin is comprised of antibodies to the diphtheria toxin. These are mostly produced in horses Infected with the diphtheria bacterium. Therefore, producing antibodies as part of the humoral immune response (Smith et al., 2017). These antibodies then bind to the AB toxin (Madigan et al., 2019) and therefore making it unable to bind to and enter the host cells.
An antibiotic and anti-toxic should be given together as they both treat the infection in two different ways once the infection occurred. Although the best way of preventing infection and therefore spread of the bacteria is via vaccination with the diphtheria toxoid.
Conclusion
Conclusively, C. Diphtheriae is a gram-positive bacterium that causes diphtheria disease. It was discovered by Edwin Klebs and Friedrich Löffler in 1884. According to history, it has mostly affected children aged below 15 and has caused more than 40,000 deaths. The number of diphtheria rates decreased in the 1920's when countries were introduced to effective immunization.
Diphtheria is a serious disease because it is linked with severe infections of the upper respiratory tract and skin ulcerations. It causes many complications such as: ear infection, airway obstruction, myocarditis and neuritis that can lead to paralysis, pneumonia, and respiratory failure.
It is transmitted from one person to another from respiratory droplets and the symptoms include fever, sore throat, and loss of appetite. The infection is identified by the pseudo membrane on the larynx, pharynx, tonsils, and nasal tissues, which is caused by the toxin.
Virulence of this disease is diphtheria exotoxin that is released from toxigenic strains of C. diphtheriae. Furthermore, the virulence of C. diphtheriae disease is associated with diphtheria toxin repressor (DtxR), because when there is an increased level of iron in the environment, the amount of toxin produced is repressed by DtxR.