The causative agent of diphtheria (Corynebacterium diphtheriae). Corynebacterium diphtheriae (diphtheria corynebacterium)
The causative agent of diphtheria belongs to the genus Corynebacterium (from the Latin coryna - club, diphthera - film). The bacteria have club-shaped thickenings at the ends. This genus includes diphtheria bacilli pathogenic for humans and non-pathogenic species - pseudodiphtheria bacilli and diphtheroids, found on the mucous membranes and skin.
The causative agents of diphtheria - Corynebacterium diphtheriae - were discovered by T. Klebs (1883) and isolated in pure form F. Leffler (1884).
Morphology. The causative agents of diphtheria are slightly curved, thin rods, 3-6 × 0.3-0.5 microns in size, with thickenings at the ends. These thickenings contain grains of volutin (Babesch-Ernst grains). Diphtheria bacteria are immobile and do not have spores or capsules. Gram positive. They are well colored with basic aniline dyes, while volutin grains are colored more intensely. Alkaline methylene blue or crystal violet are usually used for coloring. A feature of Corynebacteria diphtheria is their polymorphism; in one culture there are rods of different shapes and sizes: curved, straight, long, short, thick, sometimes coccobacteria. The location of bacteria in smears is characteristic - they are usually located in pairs at an acute or obtuse angle, in the form of splayed 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 - pseudodiphtheria bacilli and diphtheriodes are often arranged in the form of a palisade; they may have no volutin grains or may be at one end (see Fig. 4).
Cultivation. Corynebacterium diphtheria are facultative anaerobes. They grow at a temperature of 35-37° C, pH 7.4-7.8. They do not reproduce on normal nutrient media. They are cultured on media containing blood or serum.
At the end of the 19th century, the French scientist E. Roux proposed using coagulated bovine or horse serum to cultivate diphtheria bacteria, and F. Leffler recommended adding broth (25%) and 1% glucose to it. On these media, corynebacteria grow quickly, 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 pseudodiphtheria bacilli on these media.
Currently, the main growing media are Clauberg's medium (containing blood serum and potassium tellurite), Bunin's quinosol medium, Tinsdal's medium, etc. Based on the cultural and enzymatic properties of corynebacteria, diphtheria is divided into three biovars: gravis, mitis ), intermedius. Biovar gravis is usually found in the R form. On Clauberg's medium, bacteria of this biovar grow in the form of large colonies 2-3 mm, grayish-black in color (as they reduce tellurite to tellurium), and have jagged edges, which gives them the appearance of a rosette. When you touch the colony with a loop, it seems to crumble. In the broth, the bacteria of this biovar form a crumbling film and granular sediment.
Corynebacterium biovar mitis grows on Clauberg's medium in the form of small, smooth, black colonies (S-shape). In the broth they give a uniform cloudiness.
Corynebacteria biovar intermedius (intermedins) are intermediate. On Clauberg's medium, 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 possess the enzyme cystinase, which breaks down cystine to produce hydrogen sulfide. These properties are used to differentiate diphtheria pathogens from non-pathogenic representatives of this genus (Table 49).
Note. + positive reaction (breaks down); - negative reaction (does not split).
The pathogens of all three biovars break down glucose and maltose to form acid. C. gravis breaks down starch. This property distinguishes it from the other two biovars. Corynebacterium diphtheria reduces nitrates to nitrites, does not form indole, and does not decompose urea.
Corynebacterium diphtheria 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 body tissues sensitive to it. Faction A is responsible for toxic effect. The potency of diphtheria toxin cultures can be determined "in vivo" in guinea pigs sensitive to the toxin. Dim diphtheria exotoxin is the minimum lethal dose, this is the minimum amount of poison that kills a 250 g guinea pig on the 4th day.
The presence of exotoxin can also be detected “in vitro” - on a solid nutrient medium. This method is widely used in practical work. Diphtheria exotoxin is unstable. It quickly degrades 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 toxoid, which loses its toxicity, but retains the antigenic properties of the toxin. The toxins produced by different strains do not differ from each other and can be neutralized by 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 heat-labile protein antigen and a type-specific polysaccharide O-antigen. In addition, among corynebacteria, 19 phage products are distinguished, which are taken into account when identifying crops. Phagovars are used to identify the source of the disease.
Resistance to environmental factors. The causative agents of diphtheria are relatively stable. A temperature of 60° C kills them in 10-15 minutes, 100° C kills them in a minute. In film they can withstand heating up to 90° C. On rolled whey at room temperature they last up to 2 months, on children's toys - for several days. Corynebacteria tolerate low temperatures well. Diphtheria pathogens are quite resistant to drying. Disinfectants (3% phenol solution, 1% sublimate solution, 10% hydrogen peroxide solution) kill these bacteria within a few minutes.
Animal susceptibility. IN natural conditions animals do not get 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 droplets, household contact (through dishes, toys, books, towels, etc.).
Disease in humans: 1) diphtheria of the pharynx; 2) nasal diphtheria.
Less common are diphtheria of the trachea, bronchi, eyes, ears, vagina and diphtheria of damaged skin.
Pathogenesis. The entry gates are the mucous membranes of the respiratory tract and damaged skin. Once on the mucous membrane, diphtheria pathogens multiply at the site of penetration and cause tissue necrosis. A film is formed that is closely associated with the underlying tissues. Dirty gray or yellowish deposits appear on the surface of the mucosa, consisting of destroyed epithelium, fibrin, leukocytes and corynebacterium diphtheria. When removing the film with a cotton swab or spatula, the mucosal surface may bleed.
During the proliferation of Corynebacterium diphtheria, exotoxin accumulates in necrotic areas, which can lead to swelling of the mucous membrane and tissue. From the mucous membrane, swelling can spread to the larynx, bronchi and cause asphyxia. The toxin circulating in the blood selectively affects the heart muscle, adrenal glands and nervous tissue cells.
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 caused by antitoxic and antibacterial immunity. Infants do not get sick because they have passive immunity transmitted from their mother.
The presence of antitoxic immunity is judged by the Schick reaction. To set up a reaction 1 / 40 Dlm ( lethal dose Guinea pig toxin), contained in 0.2 ml of isotonic sodium chloride solution, is injected intradermally in the forearm. If there is no antitoxin in the blood, redness and swelling (up to 2 cm in diameter) appears at the injection site after 24-48 hours. If antitoxin is present, there is no swelling or redness (the antitoxin present in the blood neutralized the injected toxin).
The transferred disease leaves immunity. However, in 6-7% of cases, recurrent diseases are observed.
Prevention. Early diagnosis. Insulation. Disinfection. Identification of carriers of toxigenic diphtheria bacillus.
Specific prevention carried out by introducing toxoid. In the USSR they conduct compulsory vaccination children with the DTP vaccine is a complex vaccine that includes diphtheria and tetanus toxoid and a suspension of killed pertussis sticks. Children are vaccinated from 5-6 months with subsequent revaccination. For revaccination, a vaccine without pertussis sticks is administered.
Specific treatment. Anti-diphtheria antitoxic serum is used. The dose and frequency are determined by the attending physician, and antimicrobial drugs are also administered.
Security questions
1. What is the morphology of Corynebacterium diphtheria and what biovars are there?
2. On what media are diphtheria bacteria grown and what is the growth pattern?
3. The relationship to which carbohydrate allows one to distinguish the 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 prevention and specific treatment of diphtheria?
Microbiological examination
Purpose of the study: isolation of the pathogen for diagnosis. Identification of diphtheria bacteria carriers according to epidemiological indications. Detection of exotoxin in isolated culture.
Material for research
1. Discharge from the mucous membrane of the pharynx.
2. Discharge from the nasal mucosa.
3. Discharge from the mucous membrane of the eye.
4. Pus from the ear.
5. Discharge from the vaginal mucosa.
6. Wound discharge.
The material for research depends on the localization of the process.
For any localization of the process, be sure 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 plug, 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 no earlier than 2 hours after a meal and no earlier than 4 days after treatment with antibiotics or other antibacterial agents. 2. If the material is taken from the throat and nose, then the tubes with both swabs are labeled and tied together. Cultures are done separately and the material from each swab is examined as an independent work. 3 Material collected with a dry swab should be inoculated 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.
Progress of the study
Second day of the study
The cups are removed from the thermostat and inspected. 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 the thermostat for another 24 hours.
Third day of the study
The cups are removed from the thermostat and examined using a magnifying glass or stereoscopic microscope. If there are suspicious colonies, part of them, under the control of a stereoscopic microscope, is isolated on agar with 25% serum and on a column with Pisa medium to determine the cystinase enzyme. A test for toxigenicity is taken from another part of the colonies.
During a microscopic examination of colonies taken from Clauberg's medium, diphtheria corynebacteria lose their specificity: there is no granularity, the size changes, and the location remains the same. When they are inoculated on media with serum, the morphological specificity of diphtheria pathogens is restored.
A test for the presence of the cystinase enzyme and determination of toxigenicity are mandatory when identifying diphtheria pathogens. If the result of these experiments, carried out with part of the colonies from the Clauberg medium, is not clear enough or negative, then the experiment is repeated using the isolated pure culture.
Cystinase test. The culture under study is sown by injecting it into the center of a column of Pisa medium. If the reaction is positive, after 18-24 hours, blackening is observed during the injection, and a dark cloud forms around the black rod; blackening occurs as a result of the fact that the enzyme cystinase breaks down cystine, which is part of the Pisa 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 Pisu medium, the color of the medium does not change.
Determination of exotoxin. It is carried out by the method of diffuse precipitation in a 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 forms 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 (Martin agar produces better exotoxin). The amount of agar in the dish should be no more than 12-15 ml to maintain transparency - in a thick layer, precipitation lines are difficult to see. After the agar has hardened, apply a strip of sterile filter paper moistened with anti-diphtheria antitoxic serum.
The test culture is inoculated with “plaques”. Sowing is done using a loop. The diameter of the plaques is 0.8-1.0 cm. The distance of the plaques from the edge of the paper strips 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 nontoxigenic (Fig. 50).
Preparing strips of paper. Strips measuring 1.5x8 cm are cut from filter paper, several pieces are wrapped in paper and sterilized in an autoclave at a temperature of 120 ° C for 30 minutes. Before performing the experiment, remove one strip with sterile tweezers, place it in a sterile Petri dish and moisten it with anti-diphtheria antitoxic serum. The serum is first diluted so that 1 ml contains 500 AE (antitoxic units). The paper is moistened with 0.25 ml of serum (125 AU) and placed on the surface of the medium. Then the crops are done in the manner indicated above. All crops are placed in a thermostat. The results are recorded after 18-24 and 48 hours.
Fourth day of research
The crops are removed from the thermostat and the results are taken into account. Smears are made from a culture grown on medium with serum and stained with Loeffler's blue.
The presence in the smears of rods characteristic of their morphology, a black rod with a cloud in Pisu’s medium, and precipitation lines in agar allows us to give a preliminary answer: “Corynebacterium diphtheria has been detected.” The research continues. If there are no precipitation lines in the agar or their insufficient clarity, the toxigenicity test must be repeated with an isolated pure culture.
For final identification of the isolated culture and determination of the biovar of the pathogen, inoculation is carried out on glucose, sucrose, starch and broth with urea (to detect the urease enzyme). Sowing on media is done in the usual way.
Test for urease. The isolated culture is sown in broth with urea and an indicator (cresol red) and placed in a thermostat. After only 30-40 minutes, the result can be taken into account: when inoculating true diphtheria pathogens, the color of the medium does not change, since they do not contain urease. Pseudodiphtheria bacilli break down urea and change the indicator - the medium becomes crimson-red.
Fifth day of research
The results are recorded (Table 50).
Security questions
1. What material is examined to identify the causative agent of diphtheria?
2. How is material collected for testing for diphtheria from the throat 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 are used to determine the toxigenicity of Corynebacterium diphtheria?
1. Take wire and cotton wool from the teacher and prepare 10 tampons, install them in a cork plug, insert them into a test tube and sterilize.
Attention! Before sterilization, check whether the tampon is wound tightly enough.
2. Take sterile swabs from the teacher and collect material from each other’s throats and noses (with different swabs).
3. Study from the table. 49 properties of diphtheria pathogens and related corynebacteria.
4. Test for toxicity. Make the plaques into a loop without culture.
5. Sketch the progress of the study and the positive and negative results of the toxigenicity test.
Culture media
Tellurium Clauberg medium: first mixture - a mixture of 20 ml of lamb or horse blood and 10 ml of glycerin is prepared 1.5 months in advance. On the day of preparing the medium, 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 is added; third mixture - mix 17 ml of sheep 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 a 1% solution of potassium tellurite K 2 TeO 3, quickly everything mix and pour into cups. The medium is transparent and the color of red wine.
Wednesday Pisa. To 90 ml of molten 2% MPA (pH 7.6), add 2 ml of cystine solution (1% cystine solution 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 melted 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 2 ml tubes. Sowing is done by injection.
Wednesday Bunin. Dry quinosol medium is added to 100 ml cold water(pH 7.6-7.8), stir and heat over low heat until the agar melts (according to the instructions on the label). Then the medium is boiled for 2-3 minutes until foam forms, after which the medium is cooled to 50 ° C and 5-10 ml of sterile defibrinated blood is added. The medium is mixed and poured into Petri dishes. The prepared medium can be stored for 3-4 days at a temperature of 4-10°.
Tinsdal Wednesday. To 100 ml of 2% nutrient agar, melted and cooled to 50 ° C, add: 1) 12 ml of 1% cystine solution at 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 medium are stored for 3-4 days at 10° C.
Question 2. 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 symptoms of intoxication.
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 intensely than the cytoplasm of the cell and are easily detected when stained according to Neisser in the form of blue-black granules, while the bodies of bacteria are colored yellow-green. Gram staining does not reveal currency grains.
Drawing of a smear from a pure culture. Neisser stain Smear from pure culture.
Loeffler's alkaline blue staining
The diphtheria bacillus does not have acid resistance, is immobile, does not form spores, and has a microcapsule with a cord factor included in its composition. The cell wall contains galactose, mannose, arabinose, as well as a large number of lipids, including non-acid-stable mycolic acids.
The causative agent of diphtheria is a facultative anaerobe, heterotroph, grows at 37 o C on complex nutrient media: clotted blood serum, blood tellurite 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 skin.
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 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 uniform in cultural and biochemical properties. In accordance with the recommendations of the WHO Regional Office for Europe, the species C. diphteriae is divided into 4 biovars: gravis, mitis, intermedius, belfanti.
On tellurite medium, biovar gravis forms dry, matte, large, flat, grayish-black colonies, raised in the center. The periphery of the colony is light with radial striations 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 biovars intermedius and belfanti 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 an O-antigen (lipid and polysaccharide fractions located deep in the cell wall) and a K-antigen (a surface heat-labile protein). The O antigen is interspecies. 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 (pili, microcapsule components: cord factor, K-antigen, mycolic acids) have a protein and lipid nature, promote the adhesion of microbes in place entrance gate, prevent the phagocytosis of bacteria, have a toxic effect on the cells of the macroorganism, and destroy mitochondria.
Pathogenicity enzymes: neuraminidase, hyaluronidase, hemolysin, dermonecrotoxin. Neuraminidase splits N-acetylneuraminic acid from mucus and cell surface glycoproteins, lyase breaks it down into pyruvate and N-acetylmannosamine, and pyruvate stimulates the growth of bacteria. As a result of the action hyaluronidase the permeability of blood vessels increases and the release of plasma beyond their limits, which leads to swelling of the surrounding tissues. Dermonecrotoxin causes necrosis of cells at the site of localization of the pathogen. Plasma fibrinogen released outside the vessels contacts thrombokinase of necrotic cells of the body and turns into fibrin, which is the essence of diphtheria inflammation. Inside the diphtheria film, C. diphteriae find protection from immune system effectors and antibiotics, multiplying and forming large quantities of the main factor of pathogenicity isdiphtheria histotoxin.
Diphtheria histotoxin has a blocking effect on protein synthesis in organs most intensively supplied with blood: the cardiovascular system, myocardium, nervous system, kidneys and adrenal glands.
Epidemiology. Under natural conditions, only people who do not have resistance to the pathogen and antitoxic immunity suffer from diphtheria. The disease is widespread. The largest number of patients is observed in the second half of September, October and November. Children of preschool and primary school age are most susceptible. Among adults to group increased risk include workers in public catering and trade, schools, preschools and medical institutions.
C. diphteriae is resistant to environmental factors: in droplets of saliva adhering to dishes or toys, on door handles they can persist for up to 15 days, on environmental objects for 5.5 months, and can multiply in milk. When boiled, C. diphteriae die within 1 minute, in a 10% solution of hydrogen peroxide - after 3 minutes, in a 5% solution of carbolic acid and 50-60% alcohol - after 1 minute.
Diphtheria histotoxin is very unstable and is quickly destroyed by exposure to light, heating, and oxidation.
Pathogenesis.
Source of infection are:
1.carriers of toxigenic strains - especially dangerous are those carriers who do not have clinical manifestations of the disease, since they have antitoxic immunity.
2.patients: Among patients, those with localization of the process in the upper respiratory tract are of greatest importance. The patient is epidemiologically dangerous throughout the entire period of illness; even during the recovery period, he releases toxigenic strains into the environment.
Main mechanism of infection is aerosol. Transmission routes:
the leading role belongs to airborne droplets,
sometimes airborne dust, household contact, and also nutritional (through milk) transmission routes can occur.
Entrance gate infections serve the mucous membranes of the oropharynx ( tonsils and surrounding tissues), nose, larynx, trachea, as well as mucous membranes of the eyes and genitals, damaged skin, wound or burn surface, unhealed umbilical wound.
Most common diphtheria of the throat ( 90-95%). The incubation period lasts from 2 to 10 days. According to its pathogenesis, diphtheria belongs to toxinemic infections when the microbe remains at the entrance gate of infection, and all clinical manifestations are associated with the action of the exotoxin.
Initial stage infectious process is the adhesion of the microbe at the entrance gate. Reproducing there, the microbe secretes g istotoxin, which has a local effect on tissue cells and also enters the blood, 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 coating with a grayish or yellowish tint is formed, containing a large number of microbes that produce toxin.
A characteristic symptom 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 diphtheritic inflammation when all cells are firmly connected to each other and to the underlying connective tissue base. The fibrinous film in this case is tightly fused to the underlying tissues and cannot be removed with a tampon. When you try to do this, the mucous membrane bleeds.
Immunity. After an illness, persistent and intense humoral antitoxic immunity is formed. The duration of post-vaccination immunity is 3-5 years.
Microbiological diagnostics.
Material for research 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. Dry cotton swabs are used to collect material; if sowing will be done within 2-3 hours, when transporting the material, the swabs are moistened with a 5% glycerin solution.
Diagnostic methods:
The main diagnostic method is bacteriological. The bacteriological laboratory must give an answer after 48 hours about the presence or absence of C. diphteriae in the tests.
The material is inoculated on a nutrient medium. Suspicious colonies are selected and the isolated culture is identified:
Based on the presence of cystinase (Pizou test): the test culture is inoculated into a column of nutrient agar with cystine. The crops are incubated at 37 o C for 24 hours. C. diphteriae causes blackening of the medium along the injection as a result of the formation of lead sulfide.
For the presence of urease (Sachs test): prepare an alcoholic solution of urea and a solution of the indicator - phenol red, which are mixed before use in a ratio of 1:9 and poured into agglutination tubes. The bacteria to be studied are introduced in a loop and rubbed along the wall of the tidy up. In the positive case, after 20-30 minutes of incubation at 37 o C, the medium becomes red as a result of the breakdown of urea by urease.
The ability of C. diphteriae to produce toxin (determined by precipitation reaction in agar). To do this, a strip of filter paper impregnated with antitoxic diphtheria serum containing 5000 AE/ml is placed in a Petri dish with nutrient agar containing 15-20% horse serum, 0.3% maltose and 0.03% cystine. 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 crops are incubated at 37 0 C for 24 hours. In a positive case, at the junction of the toxin with the antitoxin in the medium, a precipitate is formed in the form of white lines - “antennae”.
To determine the toxigenicity of the causative agent of diphtheria, it can be used bioassay. The guinea pig is injected intradermally or subcutaneously with the test culture. Toxigenic cultures kill animals within 3-5 days; autopsy reveals hyperemic adrenal glands, and with intradermal infection, skin necrosis.
For bacterioscopic examination(as an independent diagnostic method 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 with Gram, the other with Neisser, the third is treated with fluorochrome - coryphosphine for fluorescent microscopy.
The presence of antitoxic immunity is judged by the Schick reaction - the reaction of neutralization of a toxin by an 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 reaction from Chic indicates the presence of antitoxins.
For accelerated detection of diphtheria toxin, both in bacterial cultures and in blood serum, the following is used: RNGA with antibody erythrocyte diagnosticum, RIA and ELISA. Molecular genetic research methods are used PCR.
Preparations for the specific treatment of diphtheria.
In order to neutralize diphtheria histotoxin, it is used specific anti-diphtheria horse purified concentrated serum, which is obtained by hyperimmunizing horses with diphtheria antitoxin.
Specific treatment with anti-diphtheria serum is started immediately if diphtheria is clinically suspected. It is necessary to choose the optimal mode of serum administration, since the antitoxin can only neutralize the toxin that is not associated with tissues. To prevent the development of anaphylactic shock, the serum is administered fractionally according to A.M. Bezredke. Administration of serum later than the 3rd day of illness is not advisable.
Designed by human anti-diphtheria immunoglobulin for intravenous administration. Its use gives fewer adverse reactions.
To suppress the proliferation of C. diphteriae at the entrance gate, antibiotics must be prescribed. 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, they use 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 reduced antigen content (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 examination allowing to identify the causative agent of diphtheria (C. diphtheriae) in the studied biomaterial.
Synonyms Russian
Culture for Loeffler's bacilli, culture for BL, culture for diphtheria bacillus.
English synonyms
Corynebacterium diphtheriae culture, Diphtheria culture.
Research method
Microbiological method.
What biomaterial can be used for research?
Throat and nose swab.
What's the right way to do research?
No preparation required.
General information about the study
Corynebacterium diphtheriae (Leffler's bacilli) are gram-positive bacteria of the genus Corynebacterium that are the causative agents of 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 symptoms of general intoxication.
At toxic form Diphtheria can also affect the heart and nervous system. In some cases, asymptomatic carriage is possible.
The diagnosis of diphtheria is based on clinical data; diphtheria culture is performed for confirmation.
What is the research used for?
- To confirm the diagnosis of diphtheria.
- For differential diagnosis diseases that occur with similar symptoms, such as tonsillitis of various origins, peritonsillar abscess, infectious mononucleosis, acute laryngotracheitis, epiglottitis, bronchial asthma.
- To evaluate the effectiveness of ongoing antibacterial therapy.
When is the study scheduled?
- If diphtheria is suspected.
- When the patient is known to have been in contact with diphtheria patients.
- After antibacterial therapy– no less than 2 weeks after finishing 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 carriage of the bacterium. At negative result culture in a patient with suspected diphtheria, the diagnosis can be confirmed when contact persons The culture result is positive, that is, the causative agent of diphtheria 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?
Previous antibacterial therapy.
Important Notes
The diagnosis of diphtheria is based on the clinical picture of the disease, so treatment should be started before laboratory confirmation of the disease is obtained. If the culture result is positive, it is necessary to examine the isolated strain of C. diphtheriae for toxigenicity.
- Culture of flora with determination of sensitivity to antibiotics
Who orders the study?
Infectious disease specialist, therapist, doctor general practice, pediatrician, ENT.
Literature
- Macgregor R.R. Corynebacterium diphtheriae. In: Principles and practice of infectious disease / G.L. Mandell, Bennett JE, 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.
Contents of the article
Corynebacterium diphtheria
First described by E. Klebs in 1983 and isolated by F. Leffler in 1984.Morphology and physiology
Corynebacterium diphtheria has a form characteristic of the entire genus. They are located at an angle to each other in the form of Roman fives. Volutin grains are revealed by staining with acetic acid blue using the Neisser method, which stains only the inclusions without affecting the cytoplasm. The diphtheria bacillus is surrounded by a microcapsule and has pili. C. diphtheriae is demanding when it comes to nutrient substrate. They need many amino acids, carbohydrates, mineral salts. They are usually cultured on clotted blood serum and potassium tellurite blood agar. On the latter medium, colonies of two types are formed: gravis - dark gray in color and mitis - black in color, which differ from each other in biochemical characteristics.Antigens
C. diphtheriae contain a K-antigen in a 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 microcapsule, with the help of which they attach to the epithelial cells of the tonsils, less often the larynx, trachea, nasal cavity, conjunctiva of the eye, vulva. Then colonization of epithelial cells occurs, which is accompanied by the appearance 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, cardiac parenchyma, kidneys, adrenal glands, and nerve ganglia. In this case, 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 genitals. 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 high level antitoxin in 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 Schick reaction - neutralization of a toxin by an antitoxin. When V40 DLM diphtheria toxin is injected into the skin of the forearm, redness and swelling appears in the absence of antitoxin in the blood. In the presence of antitoxin, the Schick reaction is negative.Ecology and epidemiology
The habitat for C. diphtheriae is people, in whose throats they are localized. Diphtheria mainly affects children. However, over the past 30 years, diphtheria has matured. In adults, diphtheria is severe and can be fatal. IN environment Diphtheria bacteria remain viable for several days because they tolerate desiccation. Infection occurs by airborne droplets and less frequently by contact.Diphtheria
Diphtheria - acute, mainly in children infectious disease, which is manifested by characteristic fibrinous inflammation at the site of localization of the pathogen and severe intoxication of the body with diphtheria exotoxin. Its causative agent is Corynebacterium diphtheriae, which belongs to the genus Corynebacterium. Before this genus there are about 20 more species of bacteria that are pathogenic for humans, animals and plants. Of these, the greatest importance is for practical medicine have the following:1. S. 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. S. jeikeium (formerly Corynebacterium JK) - causes pneumonia, endocarditis, peritonitis, infects wounds and skin.3. C. cistitidis (formerly corynebacterium group D2) - initiates the formation of stones in urinary tract and pneumonia.4. S. minutissimum - causes erythrasma, lung abscesses, endocarditis.5. S. haemolyticum - can cause tonsillitis, cellulitis, brain abscesses, osteomyelitis, chronic dermatitis.6. C. xerosis - was previously considered the causative agent of xerosis (chronic conjunctivitis), now it is classified as a saprophyte.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 research is a film from the tonsils, arches, palate, uvula, mucus from the throat and nose, less often discharge from the eye, ear, wound, vagina, affected area of the skin. At the request of the epidemiologist, washouts from toys and other objects are examined, some food products(milk, ice cream). The material must be taken before the start of etiotropic treatment on an empty stomach or 2 hours after eating. To take the material, tampons are used, dry or pre-moistened with a 5% glycerin solution, placed in a test tube and sterilized with it. The test material is taken from the oropharynx and nose with two separate swabs, trying to take it at the border of the healthy and affected area using rotational movements, without touching the swab to the mucous membrane of the cheeks, teeth and tongue, which is pressed with a spatula. During laryngoscopy, film or mucus is taken directly from the larynx. Films and mucus from the mouth and nose must be taken in all cases, even with diphtheria of rare localizations (skin, wound, eye, ear, vulva). If it is necessary to carry out an initial bacterioscopy at the request of a doctor, the material is taken with a separate (additional) swab or a part of the filmed sample is sent film, thoroughly ground between two glass slides. After collecting the material, the swabs are placed in the same test tubes, on which the number, date and time of collection, and the name of the doctor are written. They must be delivered to the laboratory no later than 3 hours after taking the material. If the sampling scheme involves taking it at the patient’s bedside, then the test tubes and dishes with cultures 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 material from a patient is carried out only at the request of a doctor and only in order to recognize necrotizing angina of Simanovsky-Plaut-Vincent (identification of spindle-shaped rods and Vincent's spirochetes, which do not grow with conventional cultivation methods). For many years microscopic examination and the identification of volutin grains, stained according to the methods of Leffler and Neisser, was the basis for the laboratory diagnosis of diphtheria and the detection of bacterial carriage. Now, due to the variability of diphtheria bacteria under the influence of antibiotics, primary microscopy of the test material is not recommended. Bacterioscopic examination is carried out to identify atypical colonies on blood-telurite media and when checking the purity of isolated cultures. Smears are stained with Gram, Leffler and Neisser. You can paint them with methyl acetate violet, toluidine blue or benthiazole and thiazine dyes. Diphtheria bacilli in smears are located at an angle, in the form of the 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. Pseudodiphtheria bacteria and diphtheroids are located in parallel (in the form of a “picket fence”) and, of course, do not have volutin grains. Babesch-Ernst grains can be detected using fluorescence microscopy when smears are stained with coryphosphine. The grains acquire an orange-red color against the background of yellow-green bacterial cell bodies.Bacteriological research
Clinical material is inoculated onto blood agar and blood telurite agar (or Clauberg II medium), poured into Petri dishes. Culture on blood agar is necessary to detect other microflora. In addition, some strains of Cdiphtheriae are sensitive to potassium tellurite, so their growth on tellurite media may be inhibited. To detect diphtheria bacteria carriage, cultures 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.Blood telurine agar
To 100 ml of 2% nutrient agar, pH 7.6, melted and cooled to 50 ° C, 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.Clauberg Wednesday 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. A glycerin mixture is prepared by adding 20 ml of sterile glycerin to 40 ml of defibrinated blood. The mixture can be stored in the refrigerator for 4 months. To prepare hemolyzed (“varnished”) blood, add 16 ml of defibrinated blood to 34 ml of sterile distilled water.Transport semi-liquid medium
To 100 ml of Hottinger's digest or meat peptone broth add 1 g of any commercial agar, set the pH to 7.6, sterilize in an autoclave at 112 ° C for 30 minutes, add 10 ml of serum and 1 ml of 2% potassium tellurite aseptically. The medium is poured into 5 ml test tubes. If possible, a more complex Emes transport medium (AMIES), modified by Stewart, is also used. Inoculation from one patient is performed on one plate, 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. You cannot sow material from several patients onto one plate. Before inoculation, the media are warmed in a thermostat for 15-20 minutes. When inoculating the test material, rub it with a swab first into a separate section of blood agar with an area of 2x1 cm, then similarly on blood telurite agar (or Clauberg II medium), while the swab is constantly turned to inoculate all the material is from it. Then, with the same swab, the remaining surfaces of the medium (half a cup) are inoculated with streaks. This plating technique produces isolated colonies (pure culture), which are used directly from the plate to determine toxigenicity and subsequent identification. Inoculated dishes or 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 there is no growth on both media, re-sample the material. Dishes with typical and suspicious colonies for C.diphtheriae are selected for further identification of the culture using all tests. Microscopy of suspicious colonies need not be performed. Colonies of diphtheria bacilli on blood agar are whitish or yellowish color, opaque, round, slightly convex, 1-2 mm in diameter. They usually have an oily consistency, although some can form brittle, hard R-colonies. On blood-telurite media, KonomiC.diphtheriae have gray , convex, with a smooth edge, viscous. After 48 hours, they acquire a dark gray or black color with a metallic sheen, equal or slightly scalloped edges, smooth or with a radially striped surface (R-form), viscous or brittle when touched with a loop. According to the structure of 48-hour colonies on telurite media and Some enzymatic characteristics 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 radial striped on the 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. Biovar intermedius 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 there is no typical growth, smears are prepared from other, doubtful colonies. If spore bacilli, cocci, yeasts, etc. are found in them, studies for 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 sowing one half of each colony on a medium to determine toxigenicity and unburned with a loop on Pisa medium, and the second half on a slanted serum agar to isolate a pure culture and preserve it until the end of laboratory diagnostics. If toxigenic and non-toxigenic varieties of C. diphtheriae grow simultaneously on a plate, it is necessary to examine the toxigenic properties of about 20 colonies in case of multiple growth of suspicious colonies, seeding material from 5-6 colonies into one plaque. When only one colony grows, it is inoculated onto a medium to determine toxigenicity and, calcining the loop, into a test tube with Pisu medium. If a transport medium was used, the hanging medium is made into dense blood-tellurite media. On the third day, when specific precipitation lines appear in an 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 dishes are incubated for another day. In the case of a negative Pisa test, the culture is identified as a species of corynebacteria. A pure culture on a slanted whey 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 cultures are recorded and a reasoned report is issued bacteriological conclusion about the isolated culture. The following methods for identifying corynebacteria are used.Toxigenicity determination in vitro
It is based on the interaction of toxin with antitoxin in an agar gel. In places of optimal quantitative ratio Toxin and antitoxin precipitate in the thickness of the agar 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, which speeds up the laboratory diagnosis of diphtheria per day. But if the test is negative, it is repeated with an isolated pure culture. To perform this test, the microbiological industry produces a special dry standard medium for determining the toxigenicity of diphtheria microbes (DTDM) and standard paper disks, soaked in antitoxic anti-diphtheria serum and dried. Paper disks are placed on the surface of the freshly prepared DTDM medium with antitoxin (no more than four per cup). At a distance of 0.5 cm from the disk, cultures are sown around it in the form of “plaques” with a diameter of 7-8 mm, 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 disks, strips of filter paper soaked in diphtheria antitoxin can be used. they are made directly in the laboratory. Paper strips cut to the specified sizes and sterilized in an autoclave at 121 ° C for 30 minutes are moistened with 0.25 ml of purified diphtheria antitoxin, which contains 500 IU in 1 ml. In this case, apply a strip of paper moistened with antitoxin to the cup with the appropriate medium and dry it by opening the cup for 15-20 minutes in a thermostat and turning it upside down. After this, cultures of “plaques” are inoculated on both sides of the strips, alternating 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 ", Kyiv (1999). For many years, the toxigenicity of diphtheria bacteria was determined by subcutaneous or intradermal injection of a culture into two Guinea pigs, one of which was injected with 100-1000 IU of antitoxic diphtheria serum the day before. Now this method bacteriological laboratories practically almost never used due to high cost and significant response delay. lately developed a very sensitive and highly specific method for determining the diphtheria toxin gene by polymerization chain reaction. It is based on determining 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, quick results (4-6 hours), and does not require the isolation of a pure culture. But it requires special equipment, expensive reagents and appropriate premises, 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 (Pizou test)
C. diphtheriae, C. ulcerans secrete the enzyme cystinase, pseudodiphtheria bacteria and other diphtheroids produce it. The isolated culture is inoculated into a medium with cystine, poured in a column into narrow test tubes. Cystinase-positive bacteria break down cystine with the release of hydrogen sulfide, which, with lead acetate included in the medium, forms lead sulfate, as a result of which the medium turns dark brown. S. diphtheriae not only causes a darkening of the environment following the course of the injection, but also forms a “cloud” around it dark brown 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 produce this enzyme. Only a few other types of corynebacteria give a positive test for urease. To perform the test, the isolated culture is sown in broth with urea. Urease decomposes urea, changes the pH of the environment, accompanied by its redness. If the enzyme is not released, the color of the broth does not change.Determination of pyrazinamidase
Determination of pyrazinamidase is carried out by hydrolysis of pyrazinamide into pyrazinoic acid and ammonium. To do this, pour 0.25 ml of sterile distilled water into a sterile test tube, in which a thick suspension of the isolated culture is prepared, then add one Rosko 598-21 diagnostic tablet. Incubate for 4 hours at 37 ° C, after which add one drop of freshly prepared 5% aqueous solution ammonium ferrous sulfate. In the presence of enzyme, the suspension turns 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 sowing a complete loop of the isolated culture into each tube of the 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
Determination of nitrate reductase is an additional test for the identification of C. belfanti and C. ulcerans, which do not produce this enzyme. The test culture is inoculated into a test tube with broth to which 0.1% KN03 is added and incubated in a thermostat for 24 hours. 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 inoculated broth, a red color appears. The medium in the control tube does not change color. To identify corynebacteria, paper indicator disks with glucose, sucrose, urea and starch from set “B” for the identification of enterobacteria (ImBio company, Nizhny Novgorod) have recently been used. A thick suspension of the test culture is prepared in 4 test tubes 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 appropriate enzymes, change color from red to yellow after 5-6 hours. When determining amylase, an indicator disk with iodine is added to a 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
Vaccinal prevention of diphtheria is carried out by administering diphtheria toxoid obtained by treating diphtheria toxin with formaldehyde. In our country, DPT - adsorbed pertussis-diphtheria-tetanus vaccine is used for vaccination. Antitoxic serum used for specific therapy, and antibiotics - for the sanitation of bacteria carriers. Antibiotics include penicillin, vancomycin, erythromycin, etc.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 corynephroma bacteria. Corynebacterium diphtheriae are straight or slightly curved rods with widened or pointed ends. Division into fracture and splitting provide a characteristic arrangement in the form of a Roman numeral V or splayed fingers, but often single sticks are found in the strokes. Large accumulations of them, which occur in smears prepared from mucus of the throat, nose, and wound discharge, have a felt-like character. The average length of their rods is 1-8 microns, width - 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 At normal concentrations they quickly die.
It is necessary to note the extremely large polymorphism of diphtheria bacilli, manifested in changes in their thickness and shape (swollen, flask-shaped, segmented, filamentous, branching). In the terminal thickenings, and sometimes in the central part, after 12 hours of culture growth, with a special coloring, Babesh-grains are found. 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 also assumptions about the need for manganese and zinc for this process.
Volutin grains are found in day-old cultures, and then the number of bacteria with the presence of grains decreases. The cytoplasm also contains nucleotide, intracytoplasmic membranes - lysosomes, vacuoles.
Bacteria are stained with all aniline dyes. When stained using the Gram method, they are positive. The Neisser method is used to stain volutin grains. When stained with this method, volutin grains, which have a high affinity for methylene blue, are persistently stained blue, and from the body of the bacterium, with additional staining with chrysoidin or bismarckbraun, methylene blue is displaced.
The causative agent of diphtheria is a heterotroph, i.e. it belongs to the group of bacteria that require for their growth organic matter. The media used for cultivation must contain amino acids as a source of carbon and nitrogen - alanine, cystine, methionine, valine, etc. In this regard, elective media for cultivation are media containing animal protein: blood, serum, ascitic fluid. On the basis of this, the classical Leffler environment was created, and then the Clauberg, Tyndall, and accumulation environments.
On Leffler's medium, colonies of diphtheria bacillus have a shiny, moist surface, smooth edges, and a yellowish color. After several days of growth, radial striations of the colonies and weakly defined 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. Optimal value The pH for the growth of diphtheria bacillus is 7.6. Corynebacterium diphtheria is often difficult to distinguish from other species of corynebacterium. To determine the species, a complex of cultural and biochemical characteristics is used.
The type of Corynebacterium diphtheria is also heterogeneous; it is divided into 3 cultural and biochemical types gravis, mitis, intermedins, into two varieties - toxigenic and nontoxigenic, a number of serological types and phagotypes.
Currently, two cultural-biochemical types circulate in most territories - gravis and mitis. The intermedins type, which used to be distinguished quite widely, has recently been rare. The most clear differentiation of types can be made by the shape of the colonies when the culture is grown on blood agar with the addition of tellurite. Colonies of the gravis type reach 1-2 mm in diameter after 48-72 hours, have wavy edges, radial striations and a flat center. Their appearance is usually compared to a daisy flower. The colonies are matte due to the ability of the bacterium to reduce tellurite, which then combines with the resulting hydrogen sulfide, gray-black in color. When growing in broth, gravis-type cultures form a crumbly film on the surface. When sown 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 tellurite blood agar grow as round, slightly convex, smooth-edged, matte black colonies. When grown in broth, they produce uniform turbidity and sediment. They do not break down starch, dextrin and glycogen.
In smears, rods of the gravis type are often short, and the mitis type is thinner and longer.
A comparative electron microscopic study of diphtheria bacilli of various biochemical types showed the presence of a three-layered structure in the gravis and mitis types. cell membrane. The shell of the intermedins type is two-layer and almost 3 times thicker. Between the cytoplasm and the membrane there are spaces filled with grains, which may be related to the exotoxin. The oblique striations of the bacteria are visible, which are created by the dividing walls between the daughter cells. The chromosomal apparatus, in the gravis and mitis types, is represented by ordinary grains with vacuoles; in the intermedins type, it is distributed throughout the cytoplasm. An electron microscope reveals a multilayered membrane, the presence of which explains why diphtheria bacilli are sometimes gram-negative.
Colonies of diphtheria bacteria come in S-, R- and SR-forms, the latter being considered intermediate. N. Morton believes that colonies of S-forms are characteristic of the mitis type, and SR-forms - of the gravis type. In addition to these main forms, there are mucoid colonies - M-forms, dwarf colonies - D-forms and gonidial colonies - L-forms. All of these 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 diphtheria bacillus. Possibility of occurrence atypical forms in laboratory conditions was confirmed by epidemiological studies.
The biochemical, morphological, physicochemical variability of diphtheria bacteria, recognized by a large number of researchers, in some cases complicates bacteriological diagnostics and forces a comprehensive study of cultures.
We divided all cultures isolated under different epidemiological conditions into 8 groups; they included all possible morphological variants of the corynebacterium representatives of interest to us:
1st group - short rods, about 2 microns long, without grains;
2nd group - short rods, about 2 microns long, but occasionally with grains;
3rd group - rods of medium size, 3-6 microns long, 0.3-0.8 microns wide, without characteristic granularity;
4th group - rods of medium size, 3-7 microns long, 0.3-0.8 microns wide, slightly curved, occasionally with grains;
5th group - rods of medium size, 3-6 microns long, 0.3-0.8 microns wide, slightly curved, granular;
6th group - long rods, 6-8 microns long, 0.3-0.6 microns wide, slightly curved, occasionally with grains;
7th group - long rods, 6-8 microns long, 0.3-0.8 microns wide, usually curved, without grains;
Group 8 - short, rough rods, about 2 microns long, about 1 microns wide, without grains.
The location of the rods was not taken into account when assigning them to groups, but usually the characteristic arrangement corresponded to the morphology.
In the 1st, 2nd, 3rd and 8th groups, which corresponded in morphology to Hoffmann's rods, the arrangement was group, parallel or in the form of single individuals, in the 4th, 5th and 6th groups, which mainly corresponded in morphology to true diphtheria bacteria, rods located at an angle or in the form of single individuals. In group 7, the rods were more often arranged randomly, intertwining with each other. In the 8th group, the rods were located in the form of single individuals.
Of the 428 cultures studied, 111, based on the totality of their characteristics, should have been classified as true diphtheria, 209 were cultures of Hoffmann’s bacilli, 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 that are morphologically close to Hoffmann's rods, they retain a positive cysteine test and 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 of these, only 9 were assigned to the 1st and 2nd morphological groups.
Cultures that were classified based on a combination of characteristics as Hoffmann's bacillus cultures had the morphology of typical diphtheria cultures in 20% of cases.
25% of the studied strains were classified as atypical cultures; their morphology corresponded to both diphtheria bacilli and Hoffmann bacilli.
Thus, biochemical and morphological properties cultures do not always coincide, and biochemical atypicality, as well as morphological, is more often observed in cultures isolated during a period of decreased incidence, and therefore a decrease in the level of carriage.
It is necessary to note 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 colonies of the same culture.
Biochemical identification of pure cultures isolated under different epidemiological conditions shows that although the morphology and biochemical properties often do not coincide, the general principle of distribution of cultures, established according to morphological data, does not change. Both when distributing crops according to morphological and biochemical data, and when fully identifying them with the inclusion serological reactions the distribution principle remains the same: atypical cultures are more common during periods of epidemic prosperity, Hoffmann bacilli are more often detected during periods 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 prosperity there are a sufficient number of carriers of toxigenic diphtheria bacilli. It should be noted that toxigenic properties cannot always be detected even in cultures isolated from patients. This indicates the need to improve the applied methods for determining the toxigenicity of crops.
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 cultures isolated in a prosperous area, according to serological data, was deeper than in disadvantaged areas. Thus, in a prosperous area, 26% of atypical cultures gave a positive agglutination reaction, in unfavorable areas - 19%.
One of the main properties of the diphtheria bacillus is the ability to form toxins. 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 performing intradermal tests for the presence of necrotic effects and 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 the 4-5th day when administered intraperitoneally. The toxin has antigenic properties that are preserved when treated with formaldehyde, 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. Intracellular synthesis of the toxin with its release through the tubules of the cell wall has been proven. The 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 a pH of 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 transform into toxigenic ones under the influence of phage. The possibility of converting nontoxigenic cultures into toxigenic ones was confirmed in experiments on single-cell cultures. The described phenomenon is called lysogenic conversion. Using temperate viruses obtained from toxigenic strains of gravis, it was possible to convert the nontoxigenic 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, a study of its role in the formation of toxigenic strains of Corynebacterium diphtheria in nature was begun. 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 common. The reasons for this are probably the lack of producers of temperate phages, the phage sensitivity of local strains is different from the reference strains, and therefore they cannot be recipients of converting phages of a known spectrum of action.
Only in part of the microbial population was it possible to convert the toxigenic properties of diphtheria bacilli under the influence of staphylococcal and streptococcal phages. In recent works, the issue of phage conversion in the epidemic process receives an even more restrained assessment. It is believed that tox+ corynephages do not play a role in the epidemic process of diphtheria independent role. Carriers of nontoxigenic bacilli can be infected with the tox+ phage only together with a toxigenic strain, and staphylococcal phages are not able to convert nontoxigenic corynebacteria. To carry out conversion towards toxigenicity in the human body, it is apparently necessary to have close contact between a carrier that has a converting phage and a carrier that secretes a strain that is lysosensitive to this phage. In addition to the ability to form toxins, the diphtheria bacterium has such pathogenicity factors as hyaluronidase, neuraminidase, deoxyribonuclease, catalase, esterase, and 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, heat-stable, surface and heat-labile antigens.
There are a number of serological typing schemes. In our country, we use the scheme proposed by V. S. Suslova and M. V. Pelevina, but it cannot provide the classification of all non-toxigenic strains. The number of serological types 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 larger, mainly due to the mitis type. From the few available literature data on patterns in the isolation of one or another serotype when various forms the infectious process and different epidemiological conditions have not been established. Along with data on the different aggressiveness of crops belonging to different serological types, there are reports that reject the connection of the serological type with the pathogenicity of crops.
It is characteristic that different serological types occur in different areas. Serological typing can be used for epidemiological analysis.
In conditions of sporadic morbidity, limited number of carriers, when the search for the source of infection is much more difficult, the phage typing method becomes important, making it possible to subdivide corynebacteria into serological and cultural variants. Marking can be carried out according to the properties of phages isolated from a culture and according to the sensitivity of the culture to specific bacteriophages. The most widely used scheme is that proposed by R. Saragea and A. Maximesco. 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: 1st group - 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 united 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 strains; strains of the gravis type were especially successfully labeled. Among the latter, belonging to one of the phage variants was established in 93%. Specific reactions with typical phages in toxigenic strains of the gravis type according to the scheme of these authors are based on infection of the 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 marking 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 a test dilution. Phages and phage variants in M.D. Krylova’s scheme are designated 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 corynebacteria of the gravis variant and a phage typing scheme for toxigenic corynebacteria of the gravis variant were developed.
