Corynebacterium

Diphtheria is an acute infectious disease predominantly of childhood, which is manifested by deep intoxication of the body with diphtheria toxin and characteristic fibrinous inflammation at the site of the pathogen. The name of the disease comes from the Greek word diphthera - skin, film, since in the breeding site of the pathogen a dense, grayish-white film forms.

The causative agent of diphtheria, Corynebacterium diphtheriae, was first discovered in 1883 by E. Klebs in slices from a film, obtained in pure culture in 1884 by F. Leffler. In 1888, E. Ru and A. Yersen discovered his ability to produce exotoxin, which plays a major role in the etiology and pathogenesis of diphtheria. The receipt in 1892 of the antitoxic serum by E. Bering and its use since 1894 for the treatment of diphtheria made it possible to significantly reduce the lethality. A successful attack on this disease began after 1923 in connection with the development of G. Rayon method of obtaining diphtheria toxoid.

The causative agent of diphtheria belongs to the genus Corynebacterium (class Actinobacteria). Morphologically, it is characterized by the fact that the cells are club-shaped thickened at the ends (Greek sogupe - mace), form branching, especially in old cultures, and contain granular inclusions.

The genus Corynebacterium includes a large number of species, which are divided into three groups.

• Corynebacteria are parasites of humans and animals and pathogenic for them.
• Corynebacteria, pathogenic for plants.
• Non-pathogenic corynebacteria. Many species of Corynebacterium are normal inhabitants of the skin, mucous throat, nasopharynx, eyes, respiratory tract, urethra and genital organs.

[1], [2], [3], [4], [5]

Morphology of corynebacteria

C. Diphtheriae - straight or slightly curved sticks of 1.0-8.0 μm in length and 0.3-0.8 μm in diameter, do not form spores and capsules. Very often they have blisters at one or both ends, often contain metachromatic granules - volute grains (polymetaphosphates), which when bluish with methylene blue get a bluish-purple color. For their detection, a special method of staining according to Neisser was proposed. In this case, the sticks are stained straw yellow, and the volute grains are dark brown, and are usually located at the poles. Corynebacterium diphtheriae is well colored with aniline dyes, Gram-positive, but in old cultures it is often discolored and has a negative Gram staining. It is characterized by pronounced polymorphism, especially in old cultures and under the influence of antibiotics. The content of G + C in DNA is about 60 mole%.

Biochemical properties of corynebacteria

Diphtheria bacillus is an aerobic or facultative anaerobic, a temperature optimum for growth of 35-37 ° C (growth boundaries of 15-40 ° C), an optimal pH of 7.6-7.8. To nutrient media is not very demanding, but it grows better on media containing serum or blood. Selective for diphtheria bacteria are coiled serum medium Py or Leffler, growth on them appears in 8-12 hours in the form of convex, the size of the pinhead of the colonies of grayish-white or yellowish-cream color. Their surface is smooth or slightly granular, on the periphery of the colony somewhat more transparent than in the center. Colonies do not merge, resulting in a culture that looks like a shagreen skin. On the broth, the growth is manifested as a uniform clouding, or the broth remains transparent, and on its surface a delicate film is formed, which gradually thickens, crumbles and flakes settles to the bottom.

A feature of diphtheria bacteria is their good growth on blood and serum media containing concentrations of potassium tellurite that suppress the growth of other bacterial species. This is due to the fact that C. Diphtheriae reconstruct potassium tellurite to metallic tellurium, which, deposited in microbial cells, gives the colonies a distinctive dark gray or black color. The use of such media increases the percentage of sowing diphtheria bacteria.

Corynebacterium diphtheriae ferment glucose, maltose, galactose to form acid without gas, but do not ferment (usually) sucrose, have cystinase, do not have urease and do not form indole. On these grounds they differ from those of coryneform bacteria (diphteroids), which are more often found on the mucous membrane of the eye (Corynebacterium xerosus) and the nasopharynx (Corynebacterium pseiidodiphtheriticum) and from other diphtheria.

In nature, there are three main variants (biotype) of diphtheria bacillus: gravis, intermedins and mitis. They differ in morphological, cultural, biochemical and other properties.

The division of diphtheria bacteria into biotypes was made taking into account the forms of diphtheria in patients with which they are allocated with the greatest frequency. The type of gravis is more often isolated from patients with severe diphtheria and causes group flares. Type mitis causes lighter and sporadic cases of diseases, and type intermedius occupies an intermediate position between them. Corynebacterium belfanti, previously attributed to the biotype mitis, is isolated in a separate, fourth, biotype. Its main difference from the biotypes gravis and mitis is the ability to restore nitrates to nitrites. Strains Corynebacterium belfanti have pronounced adhesive properties, and among them are found both toxigenic and nontoxigenic variants.

[6], [7], [8], [9], [10]

Antigenic structure of corynebacteria

Corynebacterium is very heterogeneous and mosaic. The causative agents of diphtheria of all three types revealed several dozens of somatic antigens, according to which they are divided into serotypes. In Russia, a serological classification has been adopted, according to which 11 serotypes of diphtheria bacteria are distinguished, 7 of them are primary (1-7) and 4 additional, rarely occurring serotypes (8-11). Six serotypes (1, 2, 3, 4, 5, 7) are of the gravis type, and five (6,8,9,10,11) are of the mitis type. The disadvantage of the method of serotyping is that many strains, especially nontoxigenic ones, have spontaneous agglutination or polyagglutinability.

[11]

Fototyping Corynebacterium diphtheriae

Different phage typing schemes have been proposed for the differentiation of diphtheria bacteria. According to the scheme of MD Krylova, with the help of a set of 9 phages (A, B, C, D, F, G, H, I, K), most of the toxic and non-toxic strains of the gravis type can be typed. Taking into account the sensitivity to these phages, as well as the cultural, antigenic properties and the ability to synthesize corycine (bactericidal proteins), MD Krylov singled out 3 separate groups of corynebacteria such as gravis (I-III). In each of them there are subgroups of toxigenic and nontoxigenic analogues of diphtheria causative agents.

Resistance of corynebacteria

Corynebacterium diphtheriae shows great resistance to low temperatures, but it quickly perishes at high temperature: at 60 ° C - for 15-20 min, at boiling - after 2-3 min. All disinfectants (lysol, phenol, chloramine, etc.) in the commonly used concentration destroy it in 5-10 minutes. However, the causative agent of diphtheria tolerates drying well and can remain viable for a long time in dried mucus, saliva, in dust particles. In fine-dispersed aerosol, diphtheria bacteria remain viable for 24-48 hours.

Pathogenicity factors of corynebacteria

The pathogenicity of Corynebacterium diphtheriae is determined by the presence of a number of factors.

The factors of adhesion, colonization and invasion

The structures responsible for adhesion have not been identified, but without them the diphtheria bacillus could not colonize the cells. Their role is performed by some components of the cell wall of the pathogen. Invasive properties of the causative agent are associated with hyaluronidase, neuraminidase and protease.

The toxic glycolipid contained in the cell wall of the pathogen. It is a 6,6'-diester of trehalose containing coryne-mycolic acid (C32H6403) and coryne-mycolic acid (Cs2H62O3) in equimolar ratios (trehalose-6,6'-dicorinemicolate). Glycolipid has a destructive effect on tissue cells at the site of propagation of the pathogen.

Exotoxin, which determines the pathogenicity of the pathogen and the nature of the pathogenesis of the disease. Nontoxigenic variants of C. Diphtheriae do not cause diphtheriae.

Exotoxin is synthesized as an inactive precursor - a single polypeptide chain with a m. 61 kD. Its activation is carried out by its own bacterial protease, which cuts the polypeptide into two interconnected disulfide bonds of the peptide: A (mm 21 kD) and B (m 39 kD). Peptide B performs an acceptor function - it recognizes the receptor, binds to it and forms an intramembrane channel, through which peptide A penetrates into the cell and realizes the biological activity of the toxin. Peptide A is an ADP-ribosyltransferase enzyme that provides the transfer of adenosine diphosphate ribose from NAD to one of the amino acid residues (histidine) of the EF-2 elongation protein factor. As a result of the modification, EF-2 loses its activity, and this leads to suppression of protein synthesis by ribosomes at the stage of translocation. The toxin synthesizes only such C. Diphtheriae, which carry in their chromosome the genes of the moderate converting prophage. The operon coding for the synthesis of toxin is monocistronic, it consists of 1.9 thousand pairs of nucleotides and has a toxP promoter and 3 sites: toxS, toxA and toxB. The toxS region encodes 25 amino acid residues of the signal peptide (it provides the toxin through the membrane to the periplasmic space of the bacterial cell), toxA is 193 amino acid residues of peptide A, and toxB is 342 amino acid residues of peptide B of the toxin. Loss of the cell prophage or mutation in the tox-operon makes the cell malotoxigenic. On the contrary, the lysogenization of nontoxigenic C. Diphtheriae by the converting phage turns them into toxigenic bacteria. This is proved unequivocally: toxigenicity of diphtheria bacteria depends on their lysogenization by converting tox-corynephages. The corinnephages are integrated into the chromosome of the corynebacterium by the mechanism of site-specific recombination, and strains of diphtheria bacteria can contain in their chromosomes two sites of recombination (attB), and the corynephages are integrated into each of them with the same frequency.

The genetic analysis of a number of nontoxigenic strains of diphtheria bacteria carried out with labeled DNA probes carrying fragments of the corynephag toxin has shown that their chromosomes have DNA sequences homologous to the corynephal toxin, but they either encode inactive polypeptides or are in the " silent "state, i.e., inactive. In this connection, a very important question in the epidemiological sense arises: can nontoxigenic diphtheria bacteria be transformed into toxigenic bacteria under natural conditions (in the human body), just as it occurs in vitro? The possibility of such conversion of non-toxic cultures of corynebacteria to toxigenic cultures by phage conversion was demonstrated in experiments on guinea pigs, chick embryos and white mice. However, whether this occurs during a natural epidemic process (and if so, how often), it has not been possible to establish it yet.

Due to the fact that the diphtheria toxin in the patients has a selective and specific effect on certain systems (mainly sympathetic-adrenal system, heart, vessels and peripheral nerves are affected), it is obvious that it not only inhibits protein biosynthesis in cells, but also causes other disorders of their metabolism.

To detect the toxicity of diphtheria bacteria, the following methods can be used:

• Biological tests on animals. Intracutaneous infection of guinea pigs with a filtrate of bouillon culture of diphtheria bacteria causes them necrosis at the site of administration. One minimal lethal dose of toxin (20-30 ng) kills a guinea pig weighing 250 g with a subcutaneous injection on the 4-5th day. The most characteristic manifestation of the action of the toxin is the defeat of the adrenal glands, they are enlarged and sharply hyperemic.
• Infection of chick embryos. Diphtheria toxin causes their death.
• Infection of cell cultures. Diphtheria toxin causes a distinct cytopathic effect.
• Method of solid-phase enzyme-linked immunosorbent assay using peroxidase-labeled antitoxins.
• Use of a DNA probe for direct detection of the tox-operon in the chromosome of diphtheria bacteria.

However, the most simple and common way to determine the toxicity of diphtheria bacteria is the serological method of precipitation in the gel. The essence of it is as follows. A strip of sterile 1.5 x 8 cm filter paper is moistened with an antitoxic antidiphtheria serum containing 500 AE in 1 ml and applied to the surface of the culture medium in a Petri dish. The cup is dried in a thermostat for 15-20 minutes. Test cultures are inoculated with plaques on either side of the paper. Several strains are sown on one cup, one of which, known to be toxic, serves as a control. The plates with inoculations are incubated at 37 ° C, the results taken into account after 24-48 hours. Due to counter diffusion in the gel of the antitoxin and toxin, a clear precipitation line is formed at the site of their interaction, which merges with the precipitating line of the control toxigenic strain. Strips of nonspecific precipitation (they are formed if other antimicrobial antibodies are present in small amounts besides antitoxin in a small amount) appear late, are weakly expressed and never merge with the precipitate of the control strain.

Postinfectious immunity

Strong, persistent, virtually lifelong, repeated cases of the disease are observed rarely - in 5-7% of patients who have recovered. Immunity is mainly antitoxic, antimicrobial antibodies are less important.

To assess the level of antidiphtheria immunity, Shik's test was previously widely used. To this end, 1/40 Dim of toxin for guinea pig was injected intradermally to children in a volume of 0.2 ml. In the absence of antitoxic immunity, redness and swelling of more than 1 cm in diameter appear at the site of injection 24-48 hours later. This positive reaction of Chick indicates either complete absence of the antitoxin or that its content is less than 0,001 AE / ml of blood. The negative reaction of Chick is observed when the content of antitoxin in the blood is higher than 0.03 AE / ml. If the antitoxin content is below 0.03 AE / ml, but above 0.001 AE / ml, the Shick reaction can be either positive or, sometimes, negative. In addition, the toxin itself has a pronounced allergenic property. Therefore, to determine the level of antidiphtheria immunity (the quantitative content of antitoxin), it is better to use RPGA with erythrocyte diagnosticum sensitized with diphtheria toxoid.

Epidemiology of diphtheria

The only source of infection is a person - a sick, convalescent or healthy carrier. Infection occurs by airborne, airborne dust, as well as through various items that were used in patients or healthy bacteria carrier: dishes, books, underwear, toys, etc. In case of infection of food products (milk, creams, etc.) etc.), it is possible to get infected by an alimentary route. The most massive excretion of the pathogen occurs in the acute form of the disease. However, the most epidemiologically important are people with erased, atypical forms of the disease, since they are often not hospitalized and are not immediately apparent. The diphtheria patient is contagious during the entire period of the illness and part of the recovery period. The average period of bacterial transport in convalescents varies from 2 to 7 weeks, but can last up to 3 months.

A special role in the epidemiology of diphtheria is played by healthy bacterial carriers. In conditions of sporadic morbidity, they are the main distributors of diphtheria, contributing to the preservation of the pathogen in nature. The average duration of carriage of toxigenic strains is slightly less (about 2 months) than nontoxigenic strains (about 2-3 months).

The reason for the formation of a healthy carrier of toxigenic and nontoxigenic diphtheria bacteria is not fully disclosed, since even a high level of antitoxic immunity does not always ensure the complete release of the organism from the pathogen. Perhaps, the level of antibacterial immunity is of certain importance. The carriage of toxigenic strains of diphtheria bacteria is of primary epidemiological importance.

[12], [13], [14], [15], [16], [17], [18], [19],

Symptoms of diphtheria

People of any age are susceptible to diphtheria. The causative agent can penetrate the human body through the mucous membranes of various organs or through damaged skin. Depending on the localization of the process, diphtheria of the throat, nose, larynx, ear, eye, genital organs and skin are distinguished. Possible mixed forms, for example diphtheria of throat and skin, etc. Incubation period - 2-10 days. With a clinically expressed form of diphtheria, the localization of the pathogen produces a characteristic fibrinous inflammation of the mucous membrane. The toxin produced by the pathogen first affects the epithelial cells, and then the nearby blood vessels, increasing their permeability. Exudate exudate contains fibrinogen, the coagulation of which leads to the formation on the surface of the mucous membrane of grayish-white Colors of filmy raids that are tightly welded to the underlying tissue and, when removed, cause bleeding. The consequence of the defeat of blood vessels can be the development of local edema. Particularly dangerous is the pharyngeal diphtheria, as it can cause diphtheria due to edema of the larynx and vocal cords, from which 50-60% of patients with diphtheria died as a result of asphyxia. Diphtheria toxin, entering the blood, causes a general deep intoxication. It affects primarily the cardiovascular, sympathetic-adrenal system and peripheral nerves. Thus, the symptoms of diphtheria consist of a combination of local symptoms, depending on the location of the entrance gates, and general symptoms due to toxin poisoning and manifested as adynamia, lethargy, pallor of the skin, lowering blood pressure, myocarditis, paralysis of peripheral nerves and other disorders. Diphtheria in vaccinated children, if present, occurs, as a rule, in a mild form and without complications. Mortality in the period before the application of seroterapy and antibiotics was 50-60%, now - 3-6%.

Laboratory diagnostics of diphtheria

The only method of microbiological diagnosis of diphtheria is bacteriological, with mandatory testing of the isolated culture of corynebacteria for toxigenicity. Bacteriological studies on diphtheria are carried out in three cases:

• for the diagnosis of diphtheria in children and adults with acute inflammatory processes in the area of throat, nose, nasopharynx;
• on the epidemiological indications of persons who were in contact with the source of the causative agent of diphtheria;
• persons newly admitted to orphanages, day nurseries, boarding schools, and other special institutions for children and adults, in order to identify among them possible bacterium carriers diphtheria bacillus.

The material for research is mucus from the pharynx and nose, film from tonsils or other mucous membranes, which are the entrance gate of the pathogen. Cultures are produced on tellurite whey or blood media and simultaneously on the folded serums of Pu (collapsed horse serum) or Leffler (3 parts of bovine serum + 1 part of the sugar broth), in which the growth of the corynebacteria appears after 8-12 hours. The isolated culture is identified by a set of morphological, cultural and biochemical properties, if possible, use methods of gray and phage typing. In all cases, it is necessary to check for toxicity by one of the above methods. Morphological features of corynebacteria are better studied using three methods of staining the smear: according to Gram, Neisser and methylene blue (or toluidine blue).

Treatment of diphtheria

A specific treatment for diphtheria is the use of antidiphtheria antitoxic serum containing at least 2000 IU per ml. Serum is administered intramuscularly at doses ranging from 10 000 to 400 000 IU, depending on the severity of the course of the disease. An effective method of treatment is the use of antibiotics (penicillins, tetracyclines, erythromycin, etc.) and sulfanilamide preparations. To stimulate the development of their own antitoxins, an anatoxin can be used. For the release of bacterial transport should be used those antibiotics to which this strain of corynebacteria is highly sensitive.

Specific prophylaxis of diphtheria

The main method of controlling diphtheria is a massive routine vaccination of the population. For this purpose, various variants of vaccines are used, including combined ones, ie, aimed at simultaneous creation of immunity against several pathogens. The most common vaccine in Russia was DTP. It is a suspension of pertussis bacteria adsorbed on aluminum hydroxide, killed with formalin or merthiolate (20 billion in 1 ml), and contains diphtheria toxoid in a dose of 30 flocculating units and 10 units of tetanus toxoid binding in 1 ml. Vaccinate children from 3 months of age, and then conduct a revaccination: first in 1,5-2 years, follow-up at the age of 9 and 16 years, and then every 10 years.

Thanks to the mass vaccination initiated in the USSR in 1959, the incidence of diphtheria in the country by 1966 compared with 1958 was reduced by 45 times, and its rate in 1969 was 0.7 per 100 000 population. Followed in the 80's. XX century. The reduction in the volume of vaccinations led to serious consequences. In the years 1993-1996. Russia was affected by the epidemic of diphtheria. The adults were ill, mostly not vaccinated, and the children. In 1994, almost 40 thousand patients were registered. In connection with this, mass vaccination was resumed. During this period, 132 million people were vaccinated, including 92 million adults. In 2000-2001, the coverage of children with vaccinations in the prescribed period was 96%, and the booster vaccine - 94%. Thanks to this, the incidence of diphtheria in 2001 decreased by 15 times in comparison with 1996. However, in order to bring the incidence down to single cases, it is necessary to cover at least 97-98% of children in the first year of life with vaccination and provide in the following years a massive booster dose. To achieve complete elimination of diphtheria in the coming years is unlikely to be possible due to the widespread carrier of toxigenic and nontoxigenic diphtheria bacteria. It will take some time to solve this problem.