The causative agent of diphtheria (Corynebacterium diphtheriae). Corynebacterium diphtheriae (corynebacterium diphtheria)
The causative agent of diphtheria belongs to the genus Corynebacterium (from Latin coryna - mace, diphthera - film). Bacteria have club-shaped thickenings at the ends. This genus includes diphtheria bacilli pathogenic for humans and non-pathogenic species - false diphtheria bacilli and diphtheroids found on mucous membranes and skin.
The causative agents of diphtheria - Corynebacterium diphtheriae - were discovered by T. Klebs (1883) and isolated in pure form by F. Leffler (1884).
Morphology. The causative agents of diphtheria are slightly curved, thin sticks, 3-6 × 0.3-0.5 microns in size, with thickenings at the ends. These thickenings contain volutin grains (Babesh-Ernst grains). Diphtheria bacteria are immobile, do not have spores and capsules. Gram-positive. They stain well with basic aniline dyes, while volutin grains stain more intensely. For coloring, alkaline methylene blue or crystal violet is usually used. A feature of diphtheria corynebacteria is their polymorphism; in the same culture there are sticks of various shapes and sizes: curved, straight, long, short, thick, sometimes coccobacteria. The location of bacteria in smears is characteristic - they are usually arranged in pairs at an acute or obtuse angle, in the form of spread fingers, etc. The location in smears and the presence of volutin grains is a differential diagnostic sign during microscopic examination. Non-pathogenic representatives of the genus Corynebacteria - false diphtheria bacilli and diphtheriodes are more often located in the form of a palisade, they may have no volutin grains or be at one end (see Fig. 4).
cultivation. Corynebacterium diphtheria are facultative anaerobes. Grow at a temperature of 35-37 ° C, pH 7.4-7.8. They do not reproduce on conventional nutrient media. Cultivate them on media containing blood or serum.
At the end of the 19th century, the French scientist E. Roux suggested using curdled bovine or horse serum for the cultivation of diphtheria bacteria, and F. Leffler recommended adding broth (25%) and 1% glucose to it. Corynebacteria grow rapidly on these media; within 14-18 hours they form non-merging convex cream-colored colonies (growth on a slanted medium resembles shagreen leather). However, it is impossible to differentiate diphtheria bacilli from false diphtheria on these media.
Currently, the main growing media are Clauberg's medium (containing blood serum and potassium tellurite), Bunin's quinosol medium, Tynsdal's medium, etc. Based on the cultural and enzymatic properties of corynebacteria, diphtheria is divided into three biovars: gravis (gravis), mi tis (mitis ), intermediate (intermedins). Biovar gravis is usually in the R-form. On Clauberg's medium, the bacteria of this biovar grow in the form of large colonies 2-3 mm, grayish-black in color (since they reduce tellurite to tellurium), have jagged edges, which gives them the appearance of a rosette. When you touch the colony with a loop, it seems to crumble. On the broth, the bacteria of this biovar form a crumbling film and a granular sediment.
Corynebacteria biovar mitis (mitis) grow in Clauberg's medium in the form of small, smooth colonies (S-form) of black color. On the broth, they give a uniform turbidity.
Corynebacteria biovara intermedius (intermedins) are intermediate. On Clauberg's medium, the bacteria of this biovar often grow in the form of shiny, small, black colonies (this biovar is rare).
Enzymatic properties. All three biovars of diphtheria bacteria have the enzyme cystinase, which breaks down cystine to form hydrogen sulfide. These properties are used to differentiate diphtheria pathogens from non-pathogenic representatives of this genus (Table 49).
Note. + positive reaction (splits); - negative reaction (does not split).
The causative agents of all three biovars break down glucose and maltose to form acid. C. gravis break down starch. This property distinguishes it from the other two biovars. Corynebacterium diphtheria reduce nitrates to nitrites, do not form indole, do not decompose urea.
Diphtheria Corynebacterium produces neuraminidase, hyaluronidase and other pathogenicity enzymes.
toxin formation. Virulent strains of diphtheria pathogens produce exotoxin. Chemically, it is a thermolabile protein consisting of two fractions. Fraction B fixes the toxin on sensitive tissues of the body. Fraction A is responsible for the toxic effect. The strength of diphtheria toxin cultures can be established "in vivo" in guinea pigs sensitive to this toxin. Dim diphtheria exotoxin - minimum lethal dose, this is the minimum amount of poison that kills a guinea pig weighing 250 g on the 4th day.
The presence of exotoxin can also be detected "in vitro" - on a dense nutrient medium. This method is widely used in practical work. Diphtheria exotoxin is unstable. It is rapidly destroyed under the influence of temperature, light and atmospheric oxygen. After adding formalin (0.3-0.4%) to the toxin and keeping it at a temperature of 37-38 ° C for several weeks, it turns into an anatoxin, which loses its toxicity, but retains the antigenic properties of the toxin. Toxins produced by different strains do not differ from each other and can be neutralized with diphtheria antitoxin * .
* (It has now been established that all biovars of corynebacteria can be toxigenic and non-toxigenic.)
Antigenic structure. Diphtheria bacteria have a surface thermolabile protein antigen and a type-specific polysaccharide O-antigen. In addition, 19 fagovars are distinguished among Corynebacteria, which are taken into account when identifying cultures. With the help of fagovars, the source of the disease is identified.
Environmental resistance. The causative agents of diphtheria are relatively stable. A temperature of 60 ° C kills them in 10-15 minutes, 100 ° C - in a minute. In a film, they can withstand heating up to 90 ° C. On curdled whey at room temperature, they remain for up to 2 months, on children's toys - for several days. Corynebacteria tolerate low temperatures well. The causative agents of diphtheria are quite resistant to drying. Disinfectants (3% phenol solution, 1% sublimate solution, 10% hydrogen peroxide solution) kill these bacteria within minutes.
Animal susceptibility. Under natural conditions, animals do not get sick with diphtheria. Of the experimental animals, guinea pigs and rabbits are the most susceptible. With intradermal or subcutaneous infection, they develop a picture of toxic infection with the formation of inflammation, edema, and necrosis at the injection site. Hemorrhages are observed in the adrenal glands.
Sources of the disease. Sick people and bacteria carriers.
Transmission routes. Airborne, contact-household (through dishes, toys, books, towels, etc.).
disease in humans: 1) diphtheria of the pharynx; 2) diphtheria of the nose.
Less common is diphtheria of the trachea, bronchi, eyes, ear, vagina and diphtheria of damaged skin.
Pathogenesis. The entrance gates are the mucous membranes of the respiratory tract and damaged skin. Once on the mucous membrane, diphtheria pathogens multiply at the site of introduction and cause tissue necrosis. A film is formed that is closely associated with the underlying tissues. Dirty gray or yellowish plaques appear on the surface of the mucosa, consisting of destroyed epithelium, fibrin, leukocytes and diphtheria corynebacteria. When removing the film with a cotton swab or spatula, the mucosal surface may bleed.
In the process of reproduction of Corynebacterium diphtheria, exotoxin accumulates in necrotic areas, which can lead to swelling of the mucous membrane and fiber. From the mucous membrane, edema can spread to the larynx, bronchi and cause asphyxia. The toxin circulating in the blood selectively affects the heart muscle, adrenal glands and cells of the nervous tissue.
Diphtheria is a toxic infection. The severity of the process depends on the degree of toxigenicity of the strain and on the body's defenses.
Immunity. Immunity is due to antitoxic and antibacterial immunity. Babies do not get sick, as they have passive immunity, transmitted from the mother.
The presence of antitoxic immunity is judged by the Schick reaction. To set up the reaction, 1/40 Dlm (a lethal dose of toxin for a guinea pig) contained in 0.2 ml of an isotonic sodium chloride solution is injected intradermally in the forearm. In the absence of antitoxin in the blood, redness and swelling (up to 2 cm in diameter) appear at the injection site after 24-48 hours. In the presence of antitoxin, there is no swelling and redness (the antitoxin present in the blood neutralized the injected toxin).
The transferred disease leaves immunity. However, in 6-7% of cases, repeated diseases are observed.
Prevention. Early diagnosis. Insulation. Disinfection. Identification of carriers of toxigenic diphtheria bacillus.
Specific prophylaxis carried out by the introduction of toxoid. In the USSR, compulsory vaccination of children with the DTP vaccine is carried out - this is a complex vaccine that includes diphtheria and tetanus toxoid and a suspension of killed whooping cough. Vaccinate children from 5-6 months, followed by revaccination. For revaccination, a vaccine without pertussis is administered.
Specific treatment. Apply antidiphtheria antitoxic serum. The dose and frequency rate is determined by the attending physician, antimicrobial drugs are also administered.
Control questions
1. What is the morphology of Corynebacterium diphtheria and what biovars are available?
2. On what media are diphtheria bacteria grown and what is the nature of growth?
3. Relation to what carbohydrate makes it possible to distinguish gravis biovar from other diphtheria biovars?
4. What is the route of transmission and where is the causative agent of diphtheria most often localized in a patient?
5. What are the specific prophylaxis and specific treatment for diphtheria?
Microbiological research
The purpose of the study: the isolation of the pathogen for diagnosis. Identification of bacteriocarriers of diphtheria according to epidemiological indications. Identification of exotoxin in isolated culture.
Research material
1. Detachable mucous membrane of the pharynx.
2. Discharge of the nasal mucosa.
3. Detachable mucous membrane of the eye.
4. Pus from the ear.
5. Discharge of the mucous membrane of the vagina.
6. Detachable wounds.
The material for research depends on the localization of the process.
With any localization of the process, it is imperative to examine the mucous membrane of the pharynx and nose. The material is collected with a cotton swab, for which a metal wire is used, preferably aluminum, on one end of which cotton wool is tightly wound, then the swab is mounted in a cork stopper, placed in a test tube and sterilized in a Pasteur oven at a temperature of 160 ° C 1 swab for an hour or in an autoclave at temperature 112°C.
Notes. 1. The material is collected on an empty stomach or not earlier than 2 hours after eating and not earlier than 4 days after treatment with antibiotics or other antibacterial agents. 2. If the material is taken from the pharynx and nose, then the test tubes with both swabs are inscribed and tied together. Crops are done separately and the study of the material from each swab is carried out as an independent work. 3 The material collected with a dry swab should be sown no later than 2-3 hours after collection. If it is necessary to transport the collected material, the swab is pre-moistened with a 5% solution of glycerin in an isotonic sodium chloride solution.
Basic research methods
1. Microbiological.
2. Bacterioscopic.
3. Biological.
Research progress
Second day of research
The cups are removed from the thermostat and viewed. The growth of bacteria on Clauberg's medium may be slowed down due to the presence of inhibitors in the medium. In this case, the cups are placed in a thermostat for another 24 hours.
Third day of research
The cups are removed from the thermostat, viewed with a magnifying glass or a stereoscopic microscope. In the presence of suspicious colonies, some of them, under the control of a stereoscopic microscope, are isolated on agar with 25% serum and on a column with Piso's medium to determine the cystinase enzyme. From the other part of the colonies, a toxigenicity test is put.
Microscopic examination of the colonies taken from Clauberg's medium, diphtheria corynebacteria lose their specificity: there is no granularity, the size changes, the location is preserved. When sowing them on media with serum, the morphological specificity of diphtheria pathogens is restored.
A test for the presence of the enzyme cystinase and the determination of toxigenicity are mandatory in the identification of diphtheria pathogens. If the result of these experiments, carried out with a part of the colonies from Clauberg's medium, is not clear enough or negative, then the experiment is repeated using the isolated pure culture.
Test for cystinase. The culture under study is inoculated with a prick in the center of the column of the Pisu medium. With a positive reaction, after 18-24 hours, blackening is observed along the injection, and a dark cloud forms around the black rod; blackening occurs as a result of the fact that the enzyme cystinase cleaves cystine, which is part of the Pisu medium, and the released sulfur reacts with lead acetate - black lead sulfite is formed. Diphtheroids and pseudodiphtheria bacilli do not contain the enzyme cystinase, therefore, when they grow on Piso's medium, the color of the medium does not change.
Definition of exotoxin. Carried out by the method of diffuse precipitation in the gel. The method is based on the interaction of a toxin with an antitoxin. In those areas of the agar where these components interact, a precipitate is formed in the form of rounded lines.
Method of determination: melted and cooled to 50 ° C Marten agar pH 7.8 is poured into Petri dishes (exotoxin is better produced on Marten agar). The amount of agar in the dish should be no more than 12-15 ml in order to maintain transparency - in a thick layer, the precipitation lines are poorly visible. After the agar has solidified, a strip of sterile filter paper moistened with anti-diphtheria antitoxic serum is applied.
The test culture is seeded with "plaques". Sowing is done with a loop. The diameter of the plaques is 0.8-1.0 cm. The distance of the plaques from the edge of the strips of paper is 0.5-0.7 cm; plaques of a known toxigenic strain are inoculated between two plaques of the test culture. The test culture is considered toxigenic if the precipitation lines are clear and merge with the precipitation lines of the control (toxigenic) strain. If the precipitation lines intersect with the lines of the control strain or are absent, the isolated culture is considered non-toxigenic (Fig. 50).
Preparation of paper strips. Strips of 1.5 × 8 cm in size are cut from filter paper, wrapped in several pieces in paper and sterilized in an autoclave at a temperature of 120 ° C for 30 minutes. Before setting up the experiment, one strip is taken out with sterile tweezers, placed in a sterile Petri dish and moistened with anti-diphtheria antitoxic serum. Serum is preliminarily diluted so that 1 ml contains 500 AU (antitoxic units). The paper is moistened with 0.25 ml of serum (125 AU) and placed on the surface of the medium. Then do the crops as described above. All crops are placed in a thermostat. Results are recorded after 18-24 and 48 hours.
Fourth day of research
Take out the crops from the thermostat, take into account the result. Smears are made from the culture grown on the medium with serum and stained with Loeffler's blue.
The presence in the smears of rods characteristic in morphology, a black rod with a cloud in Pisu's medium and precipitation lines in agar allows us to give a preliminary answer: "Diphtheria corynebacteria were found." The research continues. In the absence of precipitation lines in the agar or their lack of clarity, the study for toxigenicity must be repeated with an isolated pure culture.
For the final identification of the isolated culture and the determination of the biovar of the pathogen, a culture is made for glucose, sucrose, starch and urea broth (to detect the urease enzyme). Sowing on the media is done in the usual way.
Test for urease. The isolated culture is inoculated into a broth with urea and an indicator (cresol red) and placed in a thermostat. Already after 30-40 minutes, the result can be taken into account: when sowing the true pathogens of diphtheria, the color of the medium does not change, since they do not contain urease. Pseudodiphtheria sticks break down urea and change the indicator - the medium acquires a crimson-red color.
Fifth day of research
The results are recorded (Table 50).
Control questions
1. What material is examined to identify the causative agent of diphtheria?
2. How is material collected for testing for diphtheria from the pharynx and nose?
3. What should be done with the swab if the collected material needs to be transported?
4. What device is used to study colonies on Clauberg's medium?
5. What studies are carried out for the final identification of the isolated culture?
6. What methods determine the toxigenicity of corynebacterium diphtheria?
1. Take a wire and cotton wool from the teacher and prepare 10 swabs, mount them in a cork stopper, insert them into a test tube and sterilize.
Attention! Before sterilization, check if the swab is wrapped tightly enough.
2. Take sterile swabs from the teacher and take material from each other from the pharynx and nose (with different swabs).
3. Study according to the table. 49 properties of causative agents of diphtheria and corynebacteria related to them.
4. Test for toxigenicity. Make the plaques with a loop without culture.
5. Sketch the course of the study and the positive and negative results of the toxigenicity test.
Nutrient media
Tellurium Clauberg medium: the first mixture - a mixture of 20 ml of sheep or horse blood and 10 ml of glycerin is prepared 1.5 months in advance. On the day of medium preparation, two other mixtures are prepared; the second mixture - 50 ml of MPA pH 7.5 is melted and cooled to a temperature of 50 ° C, after which 2.5 ml of the first mixture are added; third mixture - mix 17 ml of sheep's blood and 33 ml of distilled water (the mixture is prepared sterile), heated in a water bath to a temperature of 50 ° C. Combine the second and third mixtures, add 4 ml of 1% potassium tellurite solution K 2 TeO 3, quickly everything mix and pour into cups. The medium is clear and has the color of red wine.
Wednesday Pisa. To 90 ml of molten 2% MPA (pH 7.6) add 2 ml of cystine solution (1% solution of cystine in 0.1 N sodium hydroxide solution), mix thoroughly and add the same volume of 0.1 N. sulfuric acid solution. The medium is sterilized for 30 minutes at a temperature of 112 ° C. To the molten and cooled to 50 ° C medium, add 1 ml of a 10% solution of lead acetate, sterilized twice with flowing steam, mix and add 9 ml of normal horse serum. The medium is sterilely poured into small test tubes of 2 ml. Sowing is done by injection.
Wednesday Bunin. Dry quinosol medium is added to 100 ml of cold water (pH 7.6-7.8), stirred and heated over low heat until the agar is melted (according to the prescription on the label). Then the medium is boiled for 2-3 minutes until foam is formed, after which the medium is cooled to 50°C and 5-10 ml of sterile defibrinated blood is added. The medium is stirred and poured into Petri dishes. The prepared medium can be stored for 3-4 days at a temperature of 4-10°C.
Tynsdale Wednesday. To 100 ml of 2% nutrient agar, melted and cooled to 50 ° C, add: 1) 12 ml of 1% cystine solution, 0.1 N. sulfuric acid solution; 2) 12 ml of 1% sodium hydroxide solution; 3) 1.8 ml of 2% potassium tellurite solution; 4) 1.8 ml of 2.5% sodium hyposulfite solution, 20 ml of normal horse or bovine serum. After adding each ingredient, the medium is thoroughly mixed. Cups with the medium are stored for 3-4 days at 10°C.
Question 2. Corynebacterium diphtheria Corynebacterium diphtheriae (genus Corynebacterium)
C. diphtheriae - rod-shaped bacteria; cause diphtheria (Greek diphtheria - skin, film) - an acute infection characterized by fibrinous inflammation in the pharynx, larynx, less often in other organs, and intoxication phenomena.
Morphological and cultural properties.
Corinebacterium diphteriae are thin, slightly curved or straight Gram-positive rods arranged at an angle to each other in the form of Roman fives. They are thickened at the ends due to the presence of grains. currency at one or both poles of the cell. Currency grains consist of polyphosphates, they perceive aniline dyes more intensively than the cell cytoplasm and are easily detected when stained according to Neisser in the form of blue-black granules, while the bodies of bacteria are stained yellow-green. When stained by Gram, currency grains are not detected.
Drawing of a smear from a pure culture. Neisser stain Smear from pure culture.
Stained with Leffler's alkaline blue
The diphtheria bacillus does not have acid resistance, is immobile, does not form spores, has a microcapsule with a cord factor included in its composition. The composition of the cell wall includes galactose, mannose, arabinose, as well as a large number of lipids, including non-acid-resistant mycolic acids.
The causative agent of diphtheria is a facultative anaerobe, a heterotroph, growing at 37 ° C on complex nutrient media: clotted blood serum, tellurite blood agar.
On elective media, after 8-14 hours, it forms dotted, convex yellowish-cream colonies with a smooth or slightly granular surface. The colonies do not merge and have the appearance of shagreen leather.
On tellurite media, the causative agent of diphtheria forms black or black-gray colonies after 24-48 hours as a result of the reduction of tellurite to metallic tellurium.
The causative agent of diphtheria has a high enzymatic activity. Differential diagnostic features C. diphteriae are:
lack of ability to ferment sucrose and decompose urea,
ability to produce the enzyme cystinase.
The causative agent of diphtheria is not homogeneous in cultural and biochemical properties. In accordance with the recommendations of the WHO Regional Office for Europe, C. diphteriae is divided into 4 biovars: gravis, mitis, intermedius, belfanti.
On tellurite medium, biovar gravis forms dry, opaque, large, flat, grayish-black colonies, raised in the center. The periphery of the colony is light with radial striation and an uneven edge. Such colonies resemble a daisy flower. Biovar mitis forms small, smooth, shiny, black, convex colonies with a smooth edge, surrounded by a zone of hemolysis. The intermedius and belfanti biovars actually belong to the mitis biovar, since they do not decompose starch, and this trait is the most stable in C. diphteriae.
Antigenic structure. C. diphteriae have O-antigen (lipid and polysaccharide fractions located deep in the cell wall) and K-antigen (surface thermolabile protein). The O antigen is cross-species. Based on the K-antigen, about 58 serovars are distinguished.
pathogenicity factors. The main pathogenicity factors of C. diphteriae are surface structures, enzymes and toxins.
Surface structures (drank, microcapsule components: cord factor, K-antigen, mycolic acids) have a protein and lipid nature, promote adhesion of microbes at the site of the entrance gate, prevent phagocytosis of bacteria, have a toxic effect on the cells of the macroorganism, and destroy mitochondria.
Enzymes of pathogenicity: neuraminidase, hyaluronidase, hemolysin, dermonecrotoxin. Neuraminidase cleaves N-acetylneuraminic acid from mucus glycoproteins and cell surfaces, lyase splits it into pyruvate and N-acetylmannosamine, and pyruvate stimulates the growth of bacteria. As a result of action hyaluronidase increases the permeability of blood vessels and the release of plasma beyond their limits, which leads to swelling of the surrounding tissues. Dermonecrotoxin causes cell necrosis at the site of the pathogen. The plasma fibrinogen that has gone beyond the limits of the vessels contacts with thrombokinase of necrotic cells of the body and turns into fibrin, which is the essence of diphtheria inflammation. Inside the diphtheria film, C. diphtheriae find protection from immune system effectors and antibiotics, multiplying, they form in large numbers the main factor of pathogenicity -diphtheria histotoxin.
Diphtheria histotoxin has a blocking effect on protein synthesis in the organs most intensively supplied with blood: the cardiovascular system, myocardium, nervous system, kidneys and adrenal glands.
Epidemiology. Under natural conditions, only a person who does not have resistance to the pathogen and antitoxic immunity suffers from diphtheria. The disease is ubiquitous. The greatest number of patients is observed in the second half of September, October and November. The most susceptible are children of preschool and primary school age. Among adults, the high-risk group includes workers in public catering and trade, schools, preschool and medical institutions.
C. diphteriae is resistant to environmental factors: in droplets of saliva stuck to dishes or toys, on door handles, they can persist for up to 15 days, on environmental objects - 5.5 months, and can multiply in milk. When boiling, C. diphteriae die within 1 min, in 10% hydrogen peroxide solution - after 3 min, in 5% carbolic acid solution and 50-60% alcohol - after 1 min.
Diphtheria histotoxin is very unstable and rapidly destroyed by light, heat, and oxidation.
Pathogenesis.
source of infection are:
1. carriers of toxigenic strains - those carriers who do not have clinical manifestations of the disease are especially dangerous, since they have antitoxic immunity.
2. Patients: Among patients, persons with localization of the process in the upper respiratory tract are of the greatest importance. The patient is epidemiologically dangerous during the entire period of illness, even during the recovery period, he releases toxigenic strains into the environment.
Main infection mechanism is aerosol. Transmission routes:
the leading role belongs to airborne,
sometimes air-dust, contact-household, and also alimentary (through milk) transmission routes can be carried out.
entrance gate infections are the mucous membranes of the oropharynx (palatine tonsils and surrounding tissues), nose, larynx, trachea, as well as the mucous membranes of the eyes and genital organs, damaged skin, wound or burn surface, unhealed umbilical wound.
Most common diphtheria pharynx ( 90-95%). The incubation period lasts from 2 to 10 days. The pathogenesis of diphtheria is toxin infections when the microbe remains at the entry gate of infection, and all clinical manifestations are associated with the action of exotoxin.
The initial stage of the infectious process is the adhesion of the microbe at the site of the entrance gate. Reproducing there, the microbe releases g istotoxin, which has a local effect on tissue cells, and also enters the bloodstream, which leads to toxinemia.
In the area of the entrance gate, an inflammatory reaction develops, which is accompanied by necrosis of epithelial cells and edema, a white plaque with a grayish or yellowish tinge is formed, containing a large number of microbes that produce the toxin.
The hallmark of diphtheria is fibrinous film:
If the mucous membrane is formed single layer epithelium(larynx, trachea, bronchi), occurs lobar inflammation, here the film is located superficially and is easily separated from the underlying tissues.
If the mucous membrane is formed stratified epithelium(oropharynx, epiglottis, vocal cords), occurs diphtheria when all cells are firmly connected to each other and to the underlying connective tissue base. The fibrinous film in this case is tightly soldered to the underlying tissues and is not removed with a swab. When you try to do this, the mucous membrane bleeds.
Immunity. After the illness, a stable and intense humoral antitoxic immunity is formed. The duration of post-vaccination immunity is 3-5 years.
Microbiological diagnostics.
Research material is a fibrinous film, mucus from the throat or nose.
The collection of material must be carried out within 3-4 hours (no later than 12 hours) from the moment the patient contacts. To take the material, dry cotton swabs are used, if the sowing is carried out within 2-3 hours, when transporting the material, the swabs are moistened with a 5% solution of glycerin.
Diagnostic methods:
The main diagnostic method is bacteriological. The bacteriological laboratory after 48 hours should give an answer about the presence or absence of C. diphteriae in the analyzes.
The material is sown on a nutrient medium. Suspicious colonies are selected and the isolated culture is identified:
According to the presence of cystinase (Pisoux test): the test culture is inoculated into a column of nutrient agar with cystine. The cultures are incubated at 37° C. for 24 hours. C. diphteriae causes the medium to turn black during the injection due to the formation of lead sulfide.
According to the presence of urease (Sachs test): an alcohol solution of urea and an indicator solution - phenol red are prepared, which are mixed before use in a ratio of 1: 9 and poured into agglutination tubes. The studied bacteria are introduced in a loop and rubbed along the wall of the tidy. In the positive case, after 20-30 minutes of incubation at 37 ° C, the medium becomes red as a result of urea cleavage by urease.
The ability of C. diphteriae to produce the toxin (determined by the agar precipitation test). To do this, in a Petri dish with nutrient agar containing 15-20% horse serum, 0.3% maltose and 0.03% cystine, a strip of filter paper soaked in antitoxic diphtheria serum containing 5000 AU/ml is placed. The cup is dried at 37 0 C for 30 minutes and the test strains are inoculated with plaques at a distance of 0.6-0.8 cm from the edge of the paper. The inoculations are incubated at 37 0 C for 24 hours. In a positive case, a precipitate is formed in the medium in the form of white lines - "antennae" at the junction of the toxin with the antitoxin.
To determine the toxigenicity of the causative agent of diphtheria can be used bioassay. A guinea pig is injected intradermally or subcutaneously with the test culture. Toxigenic cultures kill animals within 3-5 days, hyperemic adrenal glands are found at autopsy, and skin necrosis in case of intradermal infection.
For bacterioscopic examination(as an independent diagnostic method, it is rarely used due to the polymorphism of the pathogen, but can be performed at the request of a doctor) smears are prepared from the material on several glasses, one smear is stained according to Gram, the other according to Neisser, the third is treated with fluorochrome - corifosphine for luminescent microscopy.
The presence of antitoxic immunity is judged by the Schick reaction - the reaction of toxin neutralization with antitoxin. 1/40 DLM of diphtheria toxin is injected into the skin of the forearm. Redness and swelling at the injection site indicates the absence of antitoxins in the blood. A negative Schick test indicates the presence of antitoxins.
For accelerated detection of diphtheria toxin, both in bacterial cultures and in blood serum, apply: RNGA with antibody erythrocyte diagnosticum, RIA and ELISA. Of the molecular genetic research methods used PCR.
Preparations for the specific treatment of diphtheria.
In order to neutralize diphtheria histotoxin, specific anti-diphtheria equine purified concentrated serum, which is obtained by hyperimmunization of horses with diphtheria antitoxin.
Specific treatment with anti-diphtheria serum is started immediately when diphtheria is clinically suspected. It is necessary to choose the optimal mode of administration of serum, since antitoxin can only neutralize the toxin that is not associated with tissues. To prevent the development of anaphylactic shock, serum is administered fractionally according to A.M. Bezredke. The introduction of serum later than the 3rd day of illness is impractical.
Designed human diphtheria immunoglobulin for intravenous administration. Its use gives fewer side reactions.
To suppress the reproduction of C. diphteriae at the site of the entrance gate, antibiotics are mandatory. The drugs of choice are penicillin or erythromycin, or other β-lactams and macrolides.
Preparations for the specific prevention of diphtheria.
To create artificial active antitoxic immunity, apply diphtheria toxoid. The purified and concentrated drug is part of the associated vaccines:
1. adsorbed pertussis-diphtheria-tetanus vaccine (DTP vaccine),
2. adsorbed diphtheria-tetanus toxoid (ADS-toxoid),
3. adsorbed diphtheria-tetanus toxoid with a reduced content of antigens (ADS-M),
4. adsorbed diphtheria toxoid with reduced antigen content (AD-M).
Basic immunity is created in children according to the vaccination schedule. Only 95% vaccination coverage of the population guarantees the effectiveness of vaccination.
Microbiological study to identify the causative agent of diphtheria (C. diphtheriae) in the studied biomaterial.
Russian synonyms
Sowing on bacilli Leffler, sowing on BL, sowing on diphtheria bacillus.
English synonyms
Corynebacterium diphtheriae culture, Diphtheria culture.
Research method
microbiological method.
What biomaterial can be used for research?
A smear from the pharynx and nose.
How to study?
No preparation required.
General information about the study
Corynebacterium diphtheriae (Leffler's bacilli) are Gram-positive bacteria of the genus Corynebacterium that cause diphtheria and are capable of producing diphtheria toxin. The disease is transmitted by airborne droplets, the source of infection is sick people or bacteria carriers.
The incubation period averages 2-5 days. Fibrinous inflammation of the mucous membranes of the oropharynx and respiratory tract occurs with the formation of pseudomembranes and with symptoms of general intoxication.
In the toxic form of diphtheria, the heart and nervous system can also be affected. In some cases, asymptomatic carriage is possible.
The diagnosis of "diphtheria" is based on clinical findings, culture for diphtheria is performed for confirmation.
What is research used for?
- To confirm the diagnosis of diphtheria.
- For the differential diagnosis of diseases that occur with similar symptoms, such as tonsillitis of various origins, paratonsillar abscess, infectious mononucleosis, acute laryngotracheitis, epiglottitis, bronchial asthma.
- To evaluate the effectiveness of ongoing antibiotic therapy.
When is the study scheduled?
- If diphtheria is suspected.
- When the patient is known to have been in contact with patients with diphtheria.
- After antibiotic therapy - at least 2 weeks after the end of the course of antibiotics.
- In some cases, before hospitalization in a hospital (for prophylactic purposes).
What do the results mean?
Reference values: no growth.
Identification of the causative agent of diphtheria confirms the diagnosis of diphtheria or, if there are no symptoms of the disease, indicates a bacteriocarrier. With a negative culture result in a patient with suspected diphtheria, the diagnosis can be confirmed when the culture result is positive in contact persons, that is, the diphtheria causative agent is isolated.
Reasons for a positive result
- Diphtheria or asymptomatic carriage of C. diphtheriae.
Reasons for a negative result
- No diphtheria. The exception is cases when antibiotic treatment was carried out at the time of the study.
What can influence the result?
Prior antibiotic therapy.
Important Notes
The diagnosis of "diphtheria" is based on the clinical picture of the disease, so treatment should be started before receiving laboratory confirmation of the disease. With a positive culture result, it is necessary to examine the isolated strain of C. diphtheriae for toxigenicity.
- Sowing on the flora with the determination of sensitivity to antibiotics
Who orders the study?
Infectionist, therapist, general practitioner, pediatrician, ENT.
Literature
- Macgregor R.R. Corynebacterium diphtheriae. In: Principles and practice of infectious disease / G.L. Mandell, Bennett J.E., Dolin R (Eds) ; 6th ed. - Churchill Livingstone, Philadelphia, PA 2005. - 2701 p.
- Efstratiou A. Laboratory guidelines for the diagnosis of infections caused by Corynebacterium diphtheriae and C. ulceran / A. Efstratiou, R.C. Georg // Communicable disease and public health. - 1999. - Vol. 2, No. 4. - P. 250-257.
- Current approaches to the laboratory diagnosis of diphtheria / A. Efstratiou // J. Infect. Dis. - 2000. - Vol. 181 (Suppl 1). – P. S138–S145.
The content of the article
Corynebacterium diphtheria
First described by E. Klebs in 1983 and isolated by F. Leffler in 1984.Morphology and physiology
Diphtheria corynebacteria have a shape characteristic of the entire genus. They are located at an angle to each other in the form of Roman fives. Volutin grains are detected by staining with acetic blue according to the Neisser method, which stains only the inclusions without affecting the cytoplasm. The diphtheria bacillus is surrounded by a microcapsule and has a pili. C. diphtheriae are demanding on the nutrient substrate. They need many amino acids, carbohydrates, mineral salts. They are usually cultured on clotted blood serum and on blood agar with potassium tellurite. On the last medium, colonies of two types are formed: gravis - dark gray and mitis - black, which differ from each other in biochemical characteristics.Antigens
C. diphtheriae contain a K-antigen in the microcapsule, which allows them to be differentiated into serovars and a group-specific polysaccharide cell wall antigen, which gives cross-serological reactions with mycobacteria and nocardia. Pathogenicity and pathogenesis. The virulence factors of diphtheria bacteria are pili and a microcapsule, with the help of which they attach to the epitheliocytes of the tonsils, less often of the larynx, trachea, nasal cavity, conjunctiva of the eye, and vulva. Then colonization of epithelial cells occurs, which is accompanied by the onset of an inflammatory process. Toxicity is associated with the secretion of histotoxin, which consists of two subunits: a toxic polypeptide and a transport polypeptide responsible for delivering the toxic component to target cells. The formation of the first is controlled by bacterial genes, the second - by the genes of the phage that lysogenized the bacterial cell. This indicates that only lysogenic cells of C. diphtheriae can secrete histotoxin. Fixation of histotoxin occurs on the receptors of the membranes of muscle cells of the heart, heart parenchyma, kidneys, adrenal glands, and nerve ganglia. At the same time, protein synthesis on ribosomes is blocked, which ultimately leads to cell death. With diphtheria, as a rule, there is no bacteremia and septicemia due to the localization of C. diphtheriae in the cells of the larynx, where fibrinous-necrotic inflammation develops with the formation of films, lymphadenitis and edema, which can lead to asphyxia. In addition to diphtheria of the larynx, C. diphtheriae causes diphtheria of wound surfaces and genital organs. Diphtheria-like corynebacteria include the following: C. xerosis causes chronic conjunctivitis, C. ulcerans - mild forms of diphtheria-like diseases, C. pyogenes and C. haemolyticum - ulcerative necrotic pharyngitis, tonsillitis, gingivostomatitis. C. pseudodyphtheriae is a permanent inhabitant of the skin and mucous membranes.Immunity
The intensity of post-infectious immunity in diphtheria is due to the high level of antitoxin in the blood serum. Antibacterial antibodies formed during diphtheria - agglutinins, precipitins and others - do not have protective properties. The presence or absence of antitoxic immunity is judged by the Shik reaction - the neutralization of the toxin by the antitoxin. With the introduction of V40 DLM diphtheria toxin into the skin of the forearm, redness and swelling appear in the absence of antitoxin in the blood. In the presence of antitoxin, the Schick test is negative.Ecology and epidemiology
The habitat for C. diphtheriae are people in whose throat they are localized. Children are the most susceptible to diphtheria. However, over the past 30 years, diphtheria has "grown up". In adults, diphtheria is severe and can be fatal. In the environment, diphtheria bacteria remain viable for several days because they tolerate desiccation. Infection occurs by airborne droplets and less often by contact.Diphtheria
Diphtheria is an acute, predominantly childhood infectious disease, which is manifested by characteristic fibrinous inflammation at the site of the pathogen and severe intoxication of the body with diphtheria exotoxins. Its causative agent is Corynebacterium diphtheriae, belonging to the genus Corynebacterium. This genus includes about 20 more species of bacteria that are pathogenic for humans, animals and plants. Of these, the most important for practical medicine are the following: 1. C. ulcerans - can cause pharyngitis, skin lesions, it is also detected in healthy people, in dairy products and containers for their transportation, some strains are toxigenic.2. C. jeikeium (formerly Corynebacterium JK) - causes pneumonia, endocarditis, peritonitis, infects wounds, skin.3. C. cistitidis (formerly Corynebacterium group D2) - initiates the formation of stones in the urinary tract and pneumonia.4. C. minutissimum - causes erythrasma, lung abscesses, endocarditis.5. C. haemolyticum - can cause tonsillitis, cellulitis, brain abscesses, osteomyelitis, chronic dermatitis.6. C. xerosis - used to be considered the causative agent of xerosis (chronic conjunctivitis), now it is referred to as saprophytes.7. C. pseudodiphtheriticum is a saprophyte that lives on the mucous membrane of the human nasopharynx.Collection and delivery of material to the laboratory
The material for the study is a film from the tonsils, arches, palate, tongue, mucus from the throat and nose, less often discharge from the eye, ears, wounds, vagina, and the affected area of the skin. At the request of the epidemiologist, swabs from toys and other items, some food products (milk, ice cream) are examined. The material should be taken before the start of etiotropic treatment on an empty stomach or 2 hours after a meal. To take the material, swabs are used, dry or pre-moistened with a 5% glycerol solution, placed in a test tube and sterilized with it. The studied material is taken from the oropharynx and nose with two separate tampons, trying to take it on the border of the healthy and affected area with rotational movements, without touching the tampon with the mucous membranes of the cheeks, teeth and tongue, which is pressed with a spatula. With laryngoscopy, a film or mucus is taken directly from the larynx. Films and mucus from the mouth and nose are necessarily taken in all cases, even with diphtheria of rare localizations (skin, wound, eye, ear, vulva). film, carefully ground between two glass slides. After taking the material, swabs are placed in the same test tubes, on which the number, date and time of sampling, and the name of the doctor are inscribed. They must be delivered to the laboratory no later than 3 hours after taking the material. If the sampling scheme provides for occupancy at the patient's bedside, then the test tubes and dishes with crops are immediately sent to the laboratory or incubated at 37 ° C and delivered after 20-23 hours, in cold weather, in bags with heating pads.Bacterioscopic examination
A bacterioscopic examination of the material from the patient is carried out only at the request of the doctor and only in order to recognize Simanovsky-Plaut-Vincent's necrotic angina (identification of fusiform rods and Vincent's spirochetes, which do not grow with conventional cultivation methods). For many years, microscopic examination and identification of grains volutin, stained according to the methods of Leffler and Neisser, was the basis for the laboratory diagnosis of diphtheria and the detection of bacteriocarrier. Now, due to the variability of diphtheria bacteria under the influence of antibiotics, primary microscopy of the test material is not recommended. Smears are stained according to Gram, Loeffler and Neisser. You can paint them with acetic acid methyl violet, toluidine blue or benthiazol and thiazine dyes. Diphtheria bacilli in smears are located at an angle, in the form of Latin letters V, X, Y, or form clusters resembling a bunch of scattered matches. Volutin grains are located, as a rule, at the poles of microbial cells. Pseudo-diphtheria bacteria and diphtheroids are placed in parallel (in the form of a "palisade") and, of course, do not have grains of volutin. Babesh-Ernst grains can be detected using fluorescent microscopy when staining smears with corifosphine. The grains acquire an orange-red color against the background of yellow-green bodies of bacterial cells.Bacteriological research
Clinical material is inoculated on blood agar and blood-telurite agar (or Clauberg II medium) poured into Petri dishes. Blood agar culture is necessary to detect other microflora. In addition, some strains of Cdiphtheriae are sensitive to the action of potassium tellurite, so their growth on telurite media can be suppressed. To identify diphtheria bacteriocarrier, inoculations are done only on blood-telurite agar, since the inoculum may contain a small amount of diphtheria bacilli, the growth of which on non-selective media will be suppressed by other microflora. In this case, the use of a transport medium is also allowed.Telurin blood agar
To 100 ml of 2% melted and cooled to 50 ° C nutrient agar, pH 7.6, add 10-15 ml of defibrinated blood and 2 ml of 2% potassium tellurite solution. The mixture is thoroughly mixed and poured into sterile Petri dishes in a layer, 3-4 mm thick.Wednesday Clauberg II
To 100 ml of 3% nutrient agar pH 7.6, melted and cooled to 50 ° C, add 3 ml of 2% potassium tellurite solution, 10 ml of glycerol mixture and 50 ml of hemolyzed blood. The glycerin mixture is prepared by adding 20 ml of sterile glycerol to 40 ml of defibrinated blood. The mixture can be stored in the refrigerator for up to 4 months. To prepare hemolyzed ("lacquer") blood, 16 ml of defibrinated blood is added to 34 ml of sterile distilled water.Transport semi-liquid medium
1 g of any commercial agar is added to 100 ml of Hottinger's digest or meat-peptone broth, adjusted to pH 7.6, sterilized in an autoclave at 112 ° C for 30 minutes, 10 ml of serum and 1 ml of 2% potassium tellurite are added aseptically. The medium is poured into test tubes of 5 ml. If possible, a more complex Ames transport medium (AMIES) modified by Stewart is also used. Inoculation from one patient is performed on one cup, while using one half of the medium for inoculation from the oropharynx (tonsils, arches, uvula), and the second - for inoculation with another swab from the nose. If there is material to be studied from the skin, eyes, ears and other localizations, add another cup. It is impossible to sow material from several patients on one cup. Before sowing, the media are warmed in a thermostat for 15-20 minutes. When sowing the test material, rub it with a swab first into a separate area of blood agar with an area of 2x1 cm, then similarly on blood-telurite agar (or Clauberg II medium), while turning the swab all the time to sow from it all the material. Then, with the same swab, the remaining surfaces of the medium (half of the cup) are inoculated with strokes. This inoculation technique produces isolated colonies (pure culture) that are used directly from the plate for toxigenicity and subsequent identification. Inoculated dishes or test tubes with transport medium are incubated in a thermostat at 37 ° C for 20-24 hours. On the second day, the nature of the colonies is examined using a stereoscopic microscope. If growth is absent on both media, the material is resampled. Plates with typical and suspicious C. diphtheriae colonies are selected for further identification of the culture in all tests. Microscopy of suspicious colonies can be omitted. Colonies of diphtheria bacilli on blood agar are whitish or yellowish in color, opaque, round, slightly convex, 1-2 mm in diameter. They usually have an oily consistency, although some may form brittle hard R-colonies. On KonomiC. diphtheriae blood-telurite media, after 24 hours of growth, they are gray, convex, with a smooth edge, viscous. After 48 hours, they become dark gray or black with a metallic sheen, equal or slightly scalloped edges, smooth or with a radially striated surface (R-shape), viscous or brittle when touched by a loop. According to the structure of 48-hour colonies on telurite media and Some enzymatic features of the causative agent of diphtheria distinguish four cultural and biochemical variants (biovars) - gravis, mitis, belfanti, intermedius. Biovar gravis usually forms gray or black matte dry colonies, fragile, flat, smooth, 1.5-2 mm in diameter, with a radially striated surface, it is highly toxic, does not cause hemolysis, decomposes starch and glycogen. Biovars mitis and belfanti grow in the form of gray or black, round smooth convex colonies with smooth edges, 1-1.5 mm in diameter, these options are less toxic, cause hemolysis, but do not decompose starch and glycogen. Intermedius biovar forms small, gray, transparent colonies with a diameter of 0.5-1 mm, with a flat smooth surface, it is slightly toxic, does not break down starch and glycogen. If typical growth is absent, smears are prepared from other dubious colonies. If spore rods, cocci, yeast, etc. are found in them, studies on diphtheria are stopped and give a negative answer. However, it is important to remember that diphtheria bacteria that have formed atypical colonies on media with growth inhibitors (potassium tellurite) can be shortened, thickened, but retain polymorphism and characteristic location. When typical colonies grow, they immediately begin to study their toxigenicity and identification. Toxigenic properties are examined in at least 2 isolated colonies by inoculating one half of each colony on a medium for determining toxigenicity and not burned with a loop on Pisu medium, and the other half on slant serum agar to isolate a pure culture and save it until the end of laboratory diagnostics. If both toxigenic and non-toxigenic varieties of C. diphtheriae grow on the dish, it is necessary to investigate the toxigenic properties of about 20 colonies in case of multiple growth of suspicious colonies, inoculating material from 5-6 colonies into one plaque. With the growth of only one colony, it is seeded on a medium for determining toxigenicity and, by calcining a loop, into a test tube with Pisu medium. in agar gel and a positive cystinase test, the isolated culture is determined to be toxigenic C. diphtheriae. If there are no precipitation lines after 24 hours, the plates are incubated for another day. In the case of a negative Pisa test, the culture is identified as a type of corynebacteria. A pure culture on slant serum agar is sown on hydrocarbon media with glucose, sucrose, soluble starch, and samples are taken to detect urease, pyrazinamidase and nitrate reductase. On the fourth day, the results of all inoculations are recorded and a reasoned bacteriological conclusion about the isolated culture is given. Such methods of identifying corynebacteria are used.Determination of toxigenicity in vitro
It is based on the interaction of toxin with antitoxin in agar gel. In places of optimal quantitative ratio of toxin and antitoxin in the thickness of the agar, a precipitate appears in the form of thin delicate white lines ("arrows", "antennae"). This test is called the Elek-test in many countries abroad. The toxigenicity test is usually carried out with pure cultures. It can also be determined with cultures contaminated with foreign microflora; it accelerates the laboratory diagnosis of diphtheria per day. But in case of a negative test, it is repeated with an isolated pure culture. To set up this test, the microbiological industry produces a special dry standard medium for determining the toxigenicity of diphtheria microbes (VTDM) and standard paper discs soaked with antitoxic antidiphtheria serum and dried. Paper discs are applied to the surface of a freshly prepared VTDM medium with antitoxin (no more than four per cup). At a distance of 0.5 cm from the disk, cultures in the form of "plaques" with a diameter of 7-8 mm are sown around it, alternating "plaques" of the studied culture and the control strain. The results are taken into account after 18-24 and 48 years. The criterion for the specificity of precipitates is the fusion of the precipitation lines of the studied culture with the lines of the toxigenic strain. In this case, the isolated culture is considered toxigenic. In the absence of standard paper discs, filter paper strips impregnated with diphtheria antitoxin can be used. they are made directly in the laboratory. Paper strips cut to size and autoclaved at 121°C for 30 minutes are moistened with 0.25 ml of purified diphtheria antitoxin, which contains 500 IU per ml. In this case, a strip of paper moistened with antitoxin is applied to the cup with the appropriate medium, dried by opening the cup for 15-20 minutes in a thermostat and turning it upside down. After that, on both sides of the strips, the cultures are inoculated with "plaques", alternating the test and control strains. To determine the toxigenicity of the diphtheria pathogen, you can also use other media (AGV, open-hearth agar, etc.), the recipes for which are given in the "Instructions for the bacteriological diagnosis of diphtheria ", Kiev (1999). For many years, the toxigenicity of diphtheria bacteria was determined subcutaneously or intradermally by introducing a culture to two guinea pigs, one of which was injected with 100-1000 IU of antitoxic antidiphtheria serum the day before. Now bacteriological laboratories almost never use this method because of the high cost and a significant delay in the response. Recently, a very sensitive and highly specific method for determining the diphtheria toxin gene by polymerization chain reaction has been developed. It is based on the determination of the DNA region of C. diphtheriae, where the diphtheria toxin gene is localized, using specific primers. The method has advantages over the traditional determination of toxigenicity: high sensitivity, speed of obtaining results (4-6 hours), does not require the isolation of a pure culture. But its implementation requires special equipment, expensive reagents and an appropriate room, and therefore can only be carried out in a specialized laboratory. All non-toxigenic strains of diphtheria bacteria isolated from patients and bacteria carriers must be sent to the Ukrainian Center for State Sanitary and Epidemiological Surveillance (where there is such a laboratory) for the final determination of the toxigenic properties of C. diphtheriae.Determination of cystinase (Piso test)
C. diphtheriae, C. ulcerans secrete the enzyme cystinase, pseudodiphtheria bacteria and other diphtheroids produce it. The isolated culture is inoculated by injection into a medium with cystine, poured in a column into narrow test tubes. Cystine-positive bacteria break down cystine with the release of hydrogen sulfide, which, with lead acetate, which enters the medium, forms lead sulfate, as a result of which the medium turns dark brown. C. diphtheriae causes not only a darkening of the medium after the pricking course, but forms a dark brown "cloud" around it at a distance of 1 cm from the surface. The results are taken into account after 20-24 hours of incubation in a thermostat.Determination of urease (Sachse test)
Diphtheria bacteria do not form this enzyme. Only some other types of corynebacteria give a positive test for urease. To set up the sample, the isolated culture is sown in a broth with urea. Urease decomposes urea, changes the pH of the medium, accompanied by its reddening. If the enzyme is not released, there is no change in the color of the broth.Determination of pyrazinamidase
Determination of pyrazinamidase is carried out by hydrolysis of pyrazinamide to pyrazine acid and ammonium. For this, 0.25 ml of sterile distilled water is poured into a sterile test tube, in which a thick suspension of the isolated culture is prepared, then one diagnostic tablet of Rosko 598-21 is added. Incubate for 4 hours at 37°C, after which add one drop of freshly prepared 5% aqueous solution of ammonium ferrous sulfate. In the presence of the enzyme, the suspension becomes red or orange. Pathogenic corynebacteria do not secrete pyrazinamidase and therefore do not change the color of the suspension.Saccharolytic enzymes
Saccharolytic enzymes are determined by inoculating a complete loop of the isolated culture into each tube of a shortened variegated Hiss series (glucose, sucrose, soluble starch). The results are taken into account after 24 hours of incubation in a thermostat. The breakdown of starch can be delayed up to 48 hours.Determination of nitrate reductase
The determination of nitrate reductase is an additional test to identify C. belfanti and C. ulcerans that do not form this enzyme. In a test tube with broth, to which 0.1% KN03 is added, the test culture is inoculated, incubated in a thermostat for a day. Mandatory control with uninoculated medium. In the case of the presence of nitrate reductase, when 3 drops of Kasatkin's reagent are added to the seeded broth, a red color appears. The medium in the control tube does not change color. Recently, paper indicator discs with glucose, sucrose, urea and starch from the "B" set are used to identify corynebacteria for the identification of enterobacteria (firm "ImBio", Nizhny Novgorod). In 4 test tubes, a thick suspension of the culture under study is prepared, and a disk with the corresponding carbohydrate or other reagent is immersed in each of them. After incubation in a thermostat, urease release is recorded after 40-120 minutes, and saccharolytic activity is determined after 5-24 hours. In the presence of urease, the white disk with urea becomes pink-crimson, in the absence it remains white. Disks with glucose and sucrose in the presence of the corresponding enzymes change color from red to yellow after 5-6 hours. When determining amylase, an indicator disc with iodine is added to the test tube with the appropriate substrate. If there is no enzyme, a dark blue color appears; if there is, the color of the solution remains unchanged.Specific prevention and treatment
Vaccination of diphtheria is carried out with the introduction of diphtheria toxoid obtained by processing diphtheria toxin with formalin. In our country, DTP is used for vaccination - adsorbed pertussis-diphtheria-tetanus vaccine. Antitoxic serum is used for specific therapy, and antibiotics - for sanitation of bacteria carriers. Of the antibiotics, penicillin, vancomycin, erythromycin, etc. are used.Corynebacterium diphtheriae was discovered and then isolated in pure culture 100 years ago. Its final etiological significance in the occurrence of diphtheria was confirmed several years later, when a specific toxin was obtained that caused the death of animals with phenomena similar to those observed in patients with diphtheria. Corynebacterium diphtheriae belongs to the genus Corynebacterium, a group of corynebacterium bacteria. Corynebacterium diphtheriae are straight or slightly curved rods with extensions or points at the ends. Fracture division and splitting provide a characteristic arrangement in the form of a Roman numeral V or spread fingers, but single sticks are often found in strokes. Their large accumulations, which are in smears prepared from the mucus of the throat, nose, wound discharge, have a felt-like character. The average length of their sticks is 1-8 microns, the width is 0.3/0.8 microns. They are immobile and do not form spores or capsules. Corynebacterium diphtheriae is a facultative anaerobe. Diphtheria bacilli are resistant to drying. At a temperature of 60 ° C in pure cultures, they are destroyed within 45-60 minutes. In pathological products, i.e., in the presence of protein protection, they can remain viable for an hour at a temperature of 90 ° C. Low temperatures do not have a detrimental effect on diphtheria bacilli. In disinfectants of normal concentration, they quickly die.
It is necessary to note the extremely large polymorphism of diphtheria sticks, manifested in a change in their thickness and shape (swollen, flask-shaped, segmented, filiform, branching). Ernst, which are accumulations of volutin. There is evidence that volutin is a long chain inorganic polyphosphate. M. A. Peshkov suggests their metaphosphate nature. A. A. Imshanetsky believes that volutin is a by-product of metabolic processes. It is known that phosphorus is necessary for the formation of grains. There are assumptions about the need for manganese and zinc for this process.
Volutin grains are found in daily cultures, and then the number of bacteria with the presence of grains decreases. In the cytoplasm there are also nucleotides, intracytoplasmic membranes - lysosomes, vacuoles.
Bacteria are stained with all aniline dyes. When stained by the Gram method - positive. The Neisser method is used for coloring volutin grains. When stained by this method, volutin grains, which have a high affinity for methylene blue, are permanently stained blue, and methylene blue is displaced from the bacterial body with additional staining with chrysoidin or bismarckbrown.
The causative agent of diphtheria is a heterotroph, that is, it belongs to a group of bacteria that require organic matter for their growth. The media used for cultivation should contain amino acids - alanine, cystine, methionine, valine, etc. as a source of carbon and nitrogen. In this regard, media containing animal protein are elective culture media: blood, serum, ascitic fluid. On the basis of this, the classical Leffler medium was created, and then the Klauberg, Tyndall, accumulation medium.
On Leffler's medium, the colonies of diphtheria bacillus have a shiny, moist surface, smooth edges, and a yellowish color. After a few days of growth, a radial striation of the colonies and weakly expressed concentric lines appear. The diameter of the colonies reaches 4 mm. The first signs of growth appear after 6 hours in a thermostat at 36-38 °C. Growth is clearly visible 18 hours after sowing. The optimal pH value for the growth of diphtheria bacillus is 7.6. Corynebacterium diphtheria is very often difficult to distinguish from other species of Corynebacterium. A complex of cultural and biochemical features is used to determine the species.
Diphtheria Corynebacterium species is also heterogeneous, it is subdivided into 3 cultural and biochemical types gravis, mitis, intermedins, into two varieties - toxigenic and non-toxigenic, a number of serological types and phage types.
Currently, two cultural-biochemical types, gravis and mitis, circulate in most areas. The intermedins type, which used to be widely distinguished, has recently become rare. The most clear differentiation of types can be made according to the shape of the colonies when the culture is grown on blood agar with the addition of tellurite. Colonies of the gravis type after 48-72 hours reach a diameter of 1-2 mm, have wavy edges, radial striation and a flat center. Their appearance is usually compared with a daisy flower. Colonies are opaque due to the bacterium's ability to reduce tellurite, which then combines with the resulting hydrogen sulfide, gray-black in color. When grown in broth, gravis-type cultures form a crumbling film on the surface. When seeded on Hiss media with the addition of whey, they break down polysaccharides - starch, dextrin, glycogen with the formation of acid.
Cultures of the mitis type on blood agar with tellurite grow as round, slightly convex, with a smooth edge, black opaque colonies. When growing on broth, they give uniform turbidity and sediment. They do not break down starch, dextrin and glycogen.
In smears, sticks of the gravis type are often short, while those of the mitis type are thinner and longer.
A comparative electron microscopic study of diphtheria bacilli of various biochemical types showed the presence of a three-layer cell membrane in types gravis and mitis. The shell of the intermedins type is two-layered and almost 3 times thicker. Between the cytoplasm and the membrane there are spaces filled with grains, which may be related to exotoxin. Oblique striation of bacteria is visible, which is created by dividing walls between daughter cells. The chromosome apparatus, in the gravis and mitis types, is represented by ordinary grains with vacuoles, in the intermedins type it is distributed throughout the cytoplasm. In an electron microscope, a multilayered shell is visible, the presence of which explains why diphtheria bacilli are sometimes gram-negative.
Colonies of diphtheria bacteria are in S-, R- and SR-forms, the latter are considered intermediate. N. Morton believes that the colonies of S-forms are inherent in the mitis type, SR-forms - in the gravis type. In addition to these basic forms, there are colonies of the mucoid type - M-forms, dwarf colonies - D-forms and gonidial colonies - L-forms. All of them are considered forms of dissociative variability.
Diphtheria bacteria must be distinguished from diphtheroids and pseudodiphtheria bacillus.
A large number of studies are devoted to the variability of the diphtheria bacillus. The possibility of the occurrence of atypical forms in the laboratory was confirmed by the work of the epidemiological profile.
The biochemical, morphological, and physicochemical variability of the diphtheria bacterium, recognized by a large number of researchers, makes bacteriological diagnostics difficult in a number of cases and compels a comprehensive study of cultures.
We have distributed all the cultures isolated under different epidemiological conditions into 8 groups; they included all possible morphological variants of representatives of Corynebacterium that are of interest to us:
1st group - short sticks, about 2 microns long, without grains;
2nd group - short sticks, about 2 microns long, but occasionally with grains;
3rd group - sticks of medium size, 3-6 microns long, 0.3-0.8 microns wide, without characteristic granularity;
4th group - sticks of medium size, 3-7 microns long, 0.3-0.8 microns wide, slightly curved, occasionally with grains;
5th group - sticks of medium size, 3-6 microns long, 0.3-0.8 microns wide, slightly curved, granular;
6th group - long sticks, 6-8 microns long, 0.3-0.6 microns wide, slightly curved, occasionally with grains;
7th group - long sticks, 6-8 microns long, 0.3-0.8 microns wide, usually curved, without grains;
8th group - short, coarse sticks, about 2 µm long, about 1 µm wide, without grains.
The location of the rods was not taken into account during the distribution into groups, but usually the characteristic location corresponded to the morphology.
In groups 1, 2, 3 and 8, which corresponded in morphology to Hoffmann's bacilli, the arrangement was group, parallel or in the form of single individuals, in groups 4, 5 and 6, which basically corresponded in morphology to true diphtheria bacteria, bacilli located at an angle or in the form of single individuals. In the 7th group, the sticks were more often arranged randomly, intertwined with each other. In the 8th group, the rods were located in the form of single individuals.
Of the 428 cultures studied, 111, according to the combination of signs, should have been classified as true diphtheria, 209 were cultures of Hoffmann's sticks, and 108 constituted a group of atypical cultures. In cultures close to diphtheria, atypicality was manifested in a decrease in biochemical activity, sometimes in the decomposition of urea; in cultures morphologically close to Hoffmann's sticks, in maintaining a positive cysteine test, the ability to decompose one of the sugars.
Of the 111 diphtheria cultures, 81 cultures (73%) were morphologically typical, 28 cultures (27%) had the morphology of Hoffmann rods. Of the 111 diphtheria cultures, there were 20 cultures of the gravis type, and only 9 of them were assigned to the 1st and 2nd morphological groups.
The cultures, which were attributed to the cultures of the Hoffmann bacillus, in 20% of cases, had the morphology of typical diphtheria cultures.
25% of the studied strains were classified as atypical cultures; their morphology corresponded to both diphtheria bacilli and Hoffmann's bacilli.
Thus, the biochemical and morphological properties of cultures do not always coincide, and biochemical atypicality, as well as morphological, is more often observed in cultures isolated during a period of decreasing incidence, and hence a decrease in the level of carriage.
It should be noted the general decrease in the biochemical activity of crops over the past 10-15 years. An indicator of this is the delayed fermentation of sugars, which sometimes occurs on the 5-6th day, as well as the different biochemical activity of the colonies of the same culture.
Biochemical identification of pure cultures isolated under different epidemiological conditions shows that although morphology and biochemical properties often do not coincide, the general principle of distribution of cultures, established from morphological data, does not change. Both in the distribution of cultures according to morphological and biochemical data, and in their complete identification with the inclusion of serological reactions, the principle of distribution remains the same: atypical cultures are more common during the period of epidemic well-being, Hoffmann's bacilli are more often found during the period of epidemic trouble and are sown longer than true diphtheria.
The study of the toxigenic properties of isolated cultures on solid nutrient media showed that even during the period of epidemic well-being, a sufficient number of carriers of toxigenic diphtheria bacilli occurs. It should be noted that toxigenic properties are not always detectable even in cultures isolated from patients. This indicates the need to improve the methods used to determine the toxigenicity of cultures.
The results of the agglutination reaction of atypical cultures isolated under different epidemiological conditions showed the presence of the same patterns for serological properties that we noted when studying the morphology and biochemistry of cultures. The atypicality of crops isolated in a prosperous area, according to serology, was deeper than in disadvantaged areas. So, in a prosperous area, 26% of atypical cultures gave a positive agglutination reaction, in unfavorable areas - 19%.
One of the main properties of diphtheria bacillus is the ability to toxin formation. Toxinogenesis of Corynebacterium diphtheria is determined by the gene contained in the prophage, therefore, the main means of aggression - toxin formation is not associated with the bacterial chromosome.
Diphtheria toxin is a protein with a molecular weight of 6200 daltons. The strength of the toxin is determined by setting up intradermal tests for the presence of necrotic action and for the effect on susceptible animals (lethal effect). The strength of the toxin is measured using the minimum lethal dose, which is the smallest amount of toxin that can cause the death of a guinea pig weighing 250 g on days 4-5 when administered intraperitoneally. The toxin has antigenic properties that are preserved when treated with formalin, which removes its toxic properties. This made it possible to use it for the preparation of a prophylactic drug.
The toxin molecule consists of two fragments, one of which is thermostable and has enzymatic activity, and the second is thermolabile and performs a protective function. Proved intracellular synthesis of toxin with its release through the tubules of the cell wall. Synthesis of the toxin occurs when the microbe is grown in a liquid medium - meat-peptone broth with the addition of glucose, maltose and growth factors at pH 7.8-8.0.
According to recent data, diphtheria toxin is a product of viral origin. As confirmation, I. V. Chistyakova puts forward the ability of non-toxigenic corynebacteria to turn into toxigenic under the influence of the phage. The possibility of converting non-toxigenic cultures into toxigenic ones was confirmed in experiments on unicellular cultures. The described phenomenon is called lysogenic conversion. With the help of mild viruses obtained from toxigenic strains of gravis, it was possible to convert the non-toxigenic variant of Corynebacterium diphtheria gravis into a toxigenic one.
E. V. Bakulina, M. D. Krylova suggested that focal conversion may be important in the epidemic process. In this regard, the study of its role in the formation of toxigenic strains of Corynebacterium diphtheria in nature was started. The possibility of toxigenicity conversion was shown not only in phage-bacteria systems, but also in natural conditions. But among local cultures, this process, according to a number of researchers, is far from being carried out very often. The reasons for this are probably the absence of producers of temperate phages, the phage sensitivity of local strains that is different from the reference strains, and therefore they cannot be recipients of converting phages of a known spectrum of action.
Only in a part of the microbial population was the conversion of toxigenic properties in diphtheria bacilli under the action of staphylococcal and streptococcal phages successful. In the works of recent years, the issue of phage conversion in the epidemic process has received an even more restrained assessment. It is believed that tox+ corynephages do not play an independent role in the epidemic process of diphtheria. Carriers of non-toxigenic rods can be infected with tox+ phage only together with a toxigenic strain, and staphylococcal phages are not able to convert non-toxigenic corynebacteria. For the implementation of the conversion in the direction of toxigenicity in the human body, it is necessary, apparently, the presence of close contact of the carrier, which has a converting phage, with the carrier, which secretes a strain lysosensitive to this phage. In addition to the ability to form toxin, the diphtheria bacterium has such pathogenicity factors as hyaluronidase, neuraminidase, deoxyribonuclease, catalase, esterase, peroxidase. The study of extracellular metabolic products showed no differences between toxigenic and non-toxigenic diphtheria corynebacteria.
Currently, for intraspecific typing of Corynebacterium diphtheria, in addition to the biochemical method described above, serological and phage methods can be used.
The presence of serological types is due to type-specific, thermostable, surface and thermolabile antigens.
There are a number of serological typing schemes. In our country, the scheme proposed by V. S. Suslova and M. V. Pelevina is used, but it cannot ensure the classification of all non-toxigenic strains. The number of serotypes is growing. I. Ewing established the presence of 4 serological types - A, B, C and D; D. Robinson and A. Peeney 5 types - I, II, III, IV and V. L.P. Delyagina identified 2 more serological types. It is believed that the number of serological types is much greater, and mainly due to the mitis type. From the few data available in the literature, no regularities in the allocation of one or another serotype in various forms of the infectious process and various epidemiological conditions have been established. Along with data on the different aggressiveness of crops belonging to different serological types, there are reports in which the connection between the serological type and the pathogenicity of crops is rejected.
It is characteristic that different serological types are found in different territories. Serological typing can be used for epidemiological analysis.
Under conditions of sporadic morbidity, limiting the number of carriers, when it is much more difficult to search for the source of infection, the phage typing method becomes important, which makes it possible to subdivide Corynebacteria into serological and cultural variants. Marking can be carried out according to the properties of the phages isolated from the culture and according to the sensitivity of the culture to specific bacteriophages. The scheme proposed by R. Saragea and A. Maximesco is the most widely used. It allows you to label toxigenic and non-toxigenic strains of all cultural variants. With the help of 22 typical phages, cultures can be divided into 3 groups, in which 21 phage variants are combined: group 1 - toxigenic and non-toxigenic strains of the mitis type (phage variants I, la, II, III); 2nd - toxigenic and non-toxigenic strains of the intermedins type and non-toxigenic gravis (phage variants IV, V, VI, VII); 13 phage variants (from VIII to XIX) were included in the 3rd group, which combined gravis toxigenic strains.
The scheme was tested on a large number of strains isolated in Romania and obtained from museums in 14 countries. Phage typing was positive in 62% of the strains, especially strains of the gravis type were marked successfully. Among the latter, belonging to one of the phagovariants was established in 93%. Specific reactions with type phages in toxigenic strains of the gravis type according to the scheme of these authors are based on the infection of strains with various viruses.
In our country, research in the field of phage typing was carried out by M. D. Krylova. The author developed a phage labeling scheme based on the principle proposed by Williams and Rippon for typing plasmacoagulating staphylococci: the phage variant was designated by the name of the type phage that lysed it in the test dilution. Phages and phage variants in the scheme of M. D. Krylova are denoted by letters of the Latin alphabet: capital letters - phages that give confluent and semi-confluent lysis, lowercase - lysis in the form of plaques. Based on this, a modified phage typing scheme for non-toxigenic Corynebacterium gravis variant and a phage typing scheme for toxigenic Corynebacterium gravis variant were developed.
