RU2768493C1 - Method for diagnosing degree of periodontitis by determining proteinolytic activity of oral fluid microorganisms - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine and relates to a method for diagnosing the degree of periodontitis by determining the proteinolytic activity of oral fluid microorganisms, characterized by that the oral fluid is collected on an empty stomach in a sterile bottle, saliva is introduced into centrifuge tubes, chloroform is added to the sample, centrifuged and the obtained supernatant is introduced into the wells with the prepared casein-containing culture medium, incubated, the proteolysis zones are developed by adding an aqueous solution of hydrochloric acid to the agar and the result is evaluated by the size of the proteolysis zones, while the size of the proteolysis zone of 5.3 ± 0.2 mm is considered to be a natural background of the opportunistic microbiota, 8.37 ± 0.3 mm - a mild degree of periodontitis, 13.3 ± 0.4 mm - a medium degree, 16.58 ± 0.2 mm - severe periodontitis.

EFFECT: invention provides diagnosing the degree of periodontitis.

1 cl, 1 dwg, 15 ex

Description

The invention relates to medicine, namely to dentistry, microbiology and biochemistry.

Among the most important problems of modern dentistry, caries, pulpitis, inflammatory periodontal diseases occupy one of the leading places. The main cause of tooth loss in the middle and older age groups of the population of our country is periodontitis. It is known that in pathological conditions of the oral cavity and in the norm, the same microorganisms are isolated, but with altered properties and more pronounced signs of pathogenicity. Due to the large number of signs that affect the development and progression of diseases of the teeth, periodontium and oral mucosa, it is difficult to understand what processes result in the initiation and progression of the disease. Pathogenic and conditionally pathogenic microflora has many pathogenicity factors (toxins, aggression enzymes, invasions, adhesion factors, etc.), however, it is mandatory that they have proteinolytic enzymes that destroy epithelial cells, erythrocytes and other tissues of the mucous membrane and periodontium.

Prototype

In medical practice, to determine the microbiocenoses of the oral cavity, as a rule, classical bacteriological analysis is used, which requires a large number of expensive nutrient media and is long in time (at least 5 days). The test material is oral fluid in the amount of 1-2 ml. Prepare a series of dilutions and inoculation on nutrient media: Endo, Simmons, yolk-salt agar, azide agar, blood agar, lactoagar, Sabouraud, anaerobic Schedler agar, Blaurock medium, Wilson-Bler medium. Cultivation is carried out under aerobic and anaerobic conditions for 1-2 days. Identification is carried out according to a number of properties: morphological, tinctorial, cultural, biochemical, antigenic, etc. using expensive identification systems and serological reactions. It should be noted that more than 300 species of microorganisms of normal microflora live in the human oral cavity. At the same time, the ratio of anaerobes to aerobes is 100:1, and it is practically impossible to take into account all such taxonomically distant microorganisms in the classical bacteriological analysis. To identify the main pathogens, expensive methods of PCR diagnostics are used.

Screening studies for dysbacteriosis require the development of fairly fast biochemical methods that are easy to implement in practice.

An analogue, according to the authors, is a method for determining the caseinolytic (proteolytic) activity of microbes on casein-agar plates, proposed by V.M. Nikitin (Handbook of methods for biochemical express indication of microbes. Chisinau, 1986, pp. 117-119.).

When the proteinases of bacterial cultures interact with the casein contained in the agar medium, it is cleaved. When protein coagulant is added on a cloudy background of agar, zones of enlightenment around the growth of caseinolytically active microorganisms are revealed.

Investigated daily cultures of microbes on MPB or MPA; a solution of 1% casein in 1/15 M phosphate buffer pH 7.2; 2% agar for nat. solution, pH 7.2 in a test tube or a cone; developers for protein (casein): a) 10% solution of trichloroacetic acid in a vial with a ground stopper or b) glacial acetic acid 1 g, methyl alcohol 4.5 ml, dist. water 4.5 ml; dyes for protein (casein) - amido black 10 V or Kumasi bright blue R-250 in dark glass bottles with a ground glass stopper; test tubes, pipettes, Petri dishes, glass slides, punches for creating holes (wells) in agar and other laboratory equipment.

Preparation of a dye solution used to color casein protein precipitates: amido black 10 V (or Kumasi bright blue R-250) 50.0 g + ethyl alcohol 96 ° 4.5 l + glacial acetic acid 1.0 l + dist. water 4.5 l. The dye is dissolved and the solution is left overnight at room temperature, then filtered and used. The use of a dye increases the sensitivity of the method.

In 10 ml of molten and cooled to 60-65 ° C 2% agar in a test tube, add 1 ml of 1% casein solution, mix and quickly pour the contents into a sterile Petri dish or onto the surface of two glass slides. After solidification of the agar, holes with a diameter of 5-7 mm are made in it. A thick suspension of the tested microbes is introduced into each well, filling it to the brim. Cups (glass slides) with crops are placed in a thermostat at 37°C. After 6-12 hours, developer “a” or “b” is applied to the surface of the agar with samples in a volume of 4-5 ml (samples on a slide are lowered into a dish with a developer), incubated for 7-10 minutes and removed.

Registration of results is carried out visually. On a cloudy background of casein agar, with a positive result, there are clear zones with caseinolytic microbial cultures. With a negative reaction, the agar medium around the well remains uniformly cloudy. The use of dyes increases the sensitivity of the technique, since cloudy casein precipitates become more contrasting, acquiring a black-blue color, and lysis zones, on the contrary, are uncolored and light and are better detected, especially weak degrees of bacterial caseinolysis.

Some disadvantages of the proposed method are:

- poor solubility of casein in phosphate buffer,

- its uneven introduction into the nutrient medium.

As a prototype, the authors propose a biochemical express method developed in the Laboratory of Bacterial Virulence Genetics of the NIIEM named after N.I. N.F. Gamaleya (Luchshev L.I., Bondarenko V.M., Isaeva N.P., Zemskova L.N., Shakhmardanov M.Z., Gritsevskaya N.Yu. Indirect method for rapid diagnosis of intestinal dysbacteriosis in patients with salmonellosis and dysentery. Epidemiology and infectious diseases. 1996, 1: 52-54), based on the determination of the proteolytic (caseinolytic) activity of the contents of the colon, which allows a total assessment of the state of microbial biocenosis and give an approximate answer after 18 hours from the start of the study.

Testing of caseinolytic activity was carried out on Petri dishes with casein agar after 18 hours of incubation in a thermostat. Proteolysis zones were shown by adding 5% trichloroacetic acid.

The activity was considered low at a zone diameter of up to 10 mm, medium - at 11-15 mm, and high - at a diameter of proteolysis zones above 16 mm.

The disadvantages of the prototype is the lack of data on the collection of material for research, on the method of introducing casein into the nutrient medium, on the preparation of the nutrient medium for determining caseinolytic activity.

The authors propose a method for express diagnosis of the degree of periodontitis by determining the proteinolytic activity of microorganisms in the oral fluid.

For analysis, oral fluid was collected on an empty stomach in an amount of 1-2 ml in a sterile container (10 ml bottle). Before sending to the laboratory, samples can be stored for several days at +4-8°C.

The study of the proteolytic (caseinolytic) activity of the oral fluid was carried out as follows. Saliva was added to centrifuge tubes, no more than 0.05 ml (1 drop) of chloroform was added to the sample (for bacterial lysis and disinfection), centrifuged at 1500 rpm for 15 minutes, and added to the wells (no more than 9 per dish) with specially prepared nutrient medium, 20 µl. A caseinolytically active sample with a known degree of proteolysis was added to one of the control wells.

The method of preparing a nutrient medium with casein consists of the following steps. First, 100 ml of 3.5% standard nutrient agar containing yeast or fish extract and other ingredients (manufactured in Makhachkala and others) was prepared for 5 Petri dishes and brought to a boil. A 1.5% casein solution was then prepared in 0.2 N NaOH (0.15 g of casein per 10 ml of alkali). At the same time, complete dissolution of casein in an alkaline solution was achieved. After that, nutrient agar and casein solution were mixed, autoclaved at 121°C for 15 min, adjusted to pH 7.2-7.4, and poured into sterile Petri dishes. After cooling the plates on a flat surface, holes 6 mm in diameter were made in agar with a special punch or stamp. Thus, the nutrient medium with casein was ready to be introduced into the wells of the oral fluid to determine the caseinolytic (proteinolytic) activity in them (Patent for invention No. 2235324 dated August 25, 2004. Inventions. Utility models. 2004, No. 24, III part 549. (7 G01N 33/48).

After cultivation in a thermostat at 37°C for 6-18 hours, the zones of proteolysis were shown by adding 5 ml of a 5% aqueous solution of hydrochloric acid to the agar. There is no need to additionally stain the agar to reveal proteolysis zones, which reduces the number of steps and makes the method more economical.

The method allows to assess the state of microbial biocenosis, has the speed and ease of implementation, which allows it to be used in screening studies.

Microbiological examination in the oral fluid of 126 healthy people aged 17 to 60 years in 92% of cases revealed bacteria of the genus Staphylococcus, Peptostreptococcus, 88% - Streptococcus, 48% - Veillonella, Peptococcus, 44% - aerobic enterobacteria, fungi of the genus Candida , 40% - lactobacilli, 36% - micrococci, 20% - bacilli, 16% - neisseria, 12% - stomatococci, 4% - porphyromonas. The number of microorganisms averaged 4 lg CFU/ml. Signs of pathogenicity, such as hemolytic, lecithinase, protease activity, were found in 6-12% of staphylococci and streptococci.

As a result of determining the caseinolytic activity of the oral fluid of these healthy people, it was found that the diameters of the proteolysis zones averaged 5.3 ± 0.213437 mm (Fig. 1), which can be regarded as a natural background of opportunistic microbiota.

In 170 patients with periodontal pathology, smears stained by the Gram method revealed representatives of gram-positive, gram-negative microflora, convoluted forms of bacteria. Gram+ staphylococci, streptococci, rods, Gram-rods, filaments, spirochetes, leptotrichia, fusobacteria were found on the surface of the tongue and in the periodontal pocket. Moreover, the number of microorganisms in the periodontal pocket was from 2 to 100 times more than on the surface of the tongue.

Bacteriological examination of the oral fluid and the contents of the periodontal pocket of patients with periodontitis revealed that both in qualitative and quantitative composition, the microflora differed from the microflora of healthy people. In 100% of cases, microorganisms of the genus Streptococcus and Peptostreptococcus were isolated, in 96% of cases - Staphylococcus, in 68% - Micrococcus, Peptococcus and Lactobacillus, in 26% - Actinomyces, in 14-16% - Bacteroides, Porphiromonas, Bacillus, Bifidobacterium, Candida. The number of microorganisms exceeded 4 lg CFU/ml and averaged 5.8 lg CFU/ml. Pathogenicity factors were found in 24-82% of staphylococci, streptococci, peptococci, peptostreptococci, gram-positive rods. Microorganisms were isolated in associations from 4 to 8 in the studied material.

In the study of caseinolytic (proteolytic) activity according to the diameter of proteolysis zones in the oral fluid in 18 patients with mild inflammatory periodontal diseases, it was found that in individuals with limited manifestations of inflammation of the oral mucosa, the zone diameters were on average 7.6 ± 0.67 mm ( Fig. 1).

The caseinolytic activity of the oral fluid of 19 patients with periodontitis of moderate severity was on average 9.3±0.83 mm (Fig. 1). In 11 patients with severe periodontitis - 11.1±0.77 mm (Fig. 1).

Examples

1. Patient S. M. A., 29 years old. Complaints of discomfort and discomfort in the oral cavity, bleeding gums and mobility of teeth when brushing and biting off solid food. Objectively: interdental papillae and marginal gingiva are cyanotic. Periodontal pockets with a depth of no more than 3.5 mm, supra- and subgingival hard and soft dental deposits are determined. Pathological tooth mobility I degree. Hygiene index according to Green-Vermillion - 1.7. X-ray the height of the interdental septum is reduced by 1/3 of the length of the root, foci of osteoporosis, expansion of the periodontal gap.

Diagnosis: chronic generalized periodontitis, mild severity.

The diameter of the proteolysis zone is 7 mm.

2. Patient M.A.A., 28 years old. Complaints of discomfort and discomfort in the oral cavity, bleeding gums when brushing teeth and biting off solid food. Objectively: interdental papillae and marginal gingiva are cyanotic. Periodontal pockets with a depth of no more than 3.3 mm, supra- and subgingival hard and soft dental deposits are determined. Pathological tooth mobility is not observed. Hygiene index according to Green-Vermillion - 1.9. X-ray the height of the interdental septum is reduced by 1/3 of the length of the root, foci of osteoporosis, expansion of the periodontal gap.

Diagnosis: chronic generalized periodontitis, mild severity.

The diameter of the proteolysis zone is 8 mm.

3. Patient I.V.R., 35 years old. Complaints of bleeding gums when brushing teeth and biting off solid food. Objectively: interdental papillae and marginal gingiva are cyanotic. Periodontal pockets with a depth of no more than 2.7 mm, supra- and subgingival hard and soft dental deposits are determined. Pathological tooth mobility is not observed. Hygiene index according to Green-Vermillion - 2.0. X-ray the height of the interdental septum is reduced by 1/3 of the length of the root, foci of osteoporosis, expansion of the periodontal gap.

Diagnosis: chronic generalized periodontitis, mild severity.

The diameter of the proteolysis zone is 7 mm.

4. Patient Z. B.C., 37 years old. Complaints of bleeding gums when brushing teeth and biting off solid food. Objectively: interdental papillae and marginal gingiva are cyanotic. Periodontal pockets with a depth of no more than 3 mm, supra- and subgingival hard and soft dental deposits are determined. Pathological tooth mobility is not observed. Hygiene index according to Green-Vermillion - 1.8. X-ray the height of the interdental septum is reduced by 1/3 of the length of the root, foci of osteoporosis, expansion of the periodontal gap.

Diagnosis: chronic generalized periodontitis, mild severity.

The diameter of the proteolysis zone is 8 mm.

5. Patient K. A. P., 33 years old. Complaints of discomfort and discomfort in the oral cavity, bleeding gums and mobility of teeth when brushing and biting off solid food. Objectively: interdental papillae and marginal gingiva are cyanotic. Periodontal pockets with a depth of no more than 3.5 mm, supra- and subgingival hard and soft dental deposits are determined. Pathological tooth mobility I degree. Hygiene index according to Green-Vermillion - 1.7. X-ray the height of the interdental septum is reduced by 1/3 of the length of the root, foci of osteoporosis, expansion of the periodontal gap.

Diagnosis: chronic generalized periodontitis, mild severity.

The diameter of the proteolysis zone is 7 mm.

6. Patient N. E. P., 28 years old. Complaints of significant bleeding gums when eating, bad breath, mobility and displacement of teeth. Objectively: edema and hyperemia of the gums, changes in its configuration, supra- and subgingival dental deposits were detected. When probing, periodontal pockets with a depth of 5 mm are determined. Pathological tooth mobility II degree. Hygiene index according to Green-Vermillion - 2.2. X-ray the height of the interdental septum is reduced by 1/2 of the length of the root.

Diagnosis: chronic generalized periodontitis, moderate severity.

The diameter of the proteolysis zone is 9 mm.

7. Patient M. N. V., 26 years old. Complaints of significant bleeding gums when eating, bad breath, mobility and displacement of teeth. Objectively: edema and hyperemia of the gums, changes in its configuration, supra- and subgingival dental deposits were detected. When probing, periodontal pockets with a depth of 4.6 mm are determined. Pathological tooth mobility II degree. Hygiene index according to Green-Vermillion - 2. Radiographically, the height of the interdental septum is reduced by 1/2 of the length of the root.

Diagnosis: chronic generalized periodontitis, moderate severity.

The diameter of the proteolysis zone is 10 mm.

8. Patient K. P. A., 31 years old. Complaints of significant bleeding gums when eating, bad breath, mobility and displacement of teeth. Objectively: edema and hyperemia of the gums, changes in its configuration, supra- and subgingival dental deposits were detected. When probing, periodontal pockets with a depth of 5 mm are determined. Pathological tooth mobility II degree. Hygiene index according to Green-Vermillion - 2.3. X-ray the height of the interdental septum is reduced by 1/2 of the length of the root.

Diagnosis: chronic generalized periodontitis, moderate severity.

The diameter of the proteolysis zone is 10 mm.

9. Patient V.F.F., 44 years old. Complaints of significant bleeding gums when eating, bad breath, mobility and displacement of teeth. Objectively: edema and hyperemia of the gums, changes in its configuration, supra- and subgingival dental deposits were detected. When probing, periodontal pockets with a depth of 4 mm are determined. Pathological tooth mobility I degree. Hygiene index according to Green-Vermillion - 1.9. X-ray the height of the interdental septum is reduced by 1/2 of the length of the root.

Diagnosis: chronic generalized periodontitis, moderate severity.

The diameter of the proteolysis zone is 9 mm.

10. Patient F.R.V., 35 years old. Complaints of significant bleeding gums when eating, bad breath, mobility and displacement of teeth. Objectively: edema and hyperemia of the gums, changes in its configuration, supra- and subgingival dental deposits were detected. When probing, periodontal pockets with a depth of 4.4 mm are determined. Pathological tooth mobility II degree. Hygiene index according to Green-Vermillion - 2.1. X-ray the height of the interdental septum is reduced by 1/2 of the length of the root.

Diagnosis: chronic generalized periodontitis, moderate severity.

The diameter of the proteolysis zone is 9 mm.

11. Patient V. M. M., 35 years old. Complaints of bleeding gums, chewing dysfunction, misaligned teeth and bad breath. Objectively: there are significant supra- and subgingival deposits, periodontal pockets of different depths, more than 6 mm, often with purulent discharge, pathological tooth mobility reaches grade III. Hygiene index according to Green-Vermillion - 2.3. X-ray determined the destruction of bone tissue more than 1/2 of the root.

Diagnosis: chronic generalized periodontitis, severe severity.

The diameter of the proteolysis zone is 11 mm.

12. Patient M. V. A., 33 years old. Complaints of bleeding gums, chewing dysfunction, misaligned teeth and bad breath. Objectively: there are significant supra- and subgingival deposits, periodontal pockets of different depths, more than 7 mm, often with purulent discharge, pathological tooth mobility reaches grade III. Hygiene index according to Green-Vermillion - 2.4. X-ray determined the destruction of bone tissue more than 1/2 of the root.

Diagnosis: chronic generalized periodontitis, severe severity.

The diameter of the proteolysis zone is 11 mm.

13. Patient Sh. S.V., 24 years old. Complaints of bleeding gums, chewing dysfunction, misaligned teeth and bad breath. Objectively: there are significant supra- and subgingival deposits, periodontal pockets of different depths, more than 8 mm, often with purulent discharge, pathological tooth mobility reaches grade IV. Hygiene index according to Green-Vermillion - 2.5. X-ray determined the destruction of bone tissue more than 1/2 of the root.

Diagnosis: chronic generalized periodontitis, severe severity.

The diameter of the proteolysis zone is 12 mm.

14. Patient Sh. M.A., 35 years old. Complaints of bleeding gums, chewing dysfunction, misaligned teeth and bad breath. Objectively: significant supra- and subgingival dental deposits are revealed, probing reveals periodontal pockets 7.2 mm deep, often with purulent discharge. Pathological tooth mobility reaches IV degree. Hygiene index according to Green-Vermillion - 2.3. X-ray determined the destruction of bone tissue more than 1/2 of the root.

Diagnosis: chronic generalized periodontitis, severe severity.

The diameter of the proteolysis zone is 11 mm.

15. Patient C. M. V., 39 years old. Complaints of bleeding gums, chewing dysfunction, misaligned teeth and bad breath. Objectively: significant supra- and subgingival dental deposits were revealed, probing revealed periodontal pockets 7.4 mm deep, often with purulent discharge. Pathological tooth mobility reaches IV degree. Hygiene index according to Green-Vermillion - 2.3. X-ray determined the destruction of bone tissue more than 1/2 of the root.

Diagnosis: chronic generalized periodontitis, severe severity.

The diameter of the proteolysis zone is 11 mm.

As a result of microbiological studies, it was found that the microflora of the oral fluid and periodontal pocket of patients with periodontal pathology differs in species composition from the microflora of healthy people. In patients, along with normal representatives, proteolytically active gram-positive and gram-negative anaerobic prevotella, leptotrichia, spirochetes, etc. were detected. The number of microorganisms in patients increases by 1-3 lg CFU / ml, while bacteria with such pathogenicity factors as hemolytic, lecithinase, proteolytic, etc. Thus, the involvement of the association of opportunistic microflora in the development of inflammatory periodontal diseases has been established and a relationship has been revealed between the presence of pathogenic and opportunistic microflora in the oral fluid and the degree of its proteolytic (caseinolytic) activity.

Claims (1)

A method for diagnosing the degree of periodontitis by determining the proteinolytic activity of oral fluid microorganisms, characterized in that the oral fluid is collected on an empty stomach in an amount of 1-2 ml in a sterile 10 ml vial, saliva is added to centrifuge tubes, no more than 0.05 ml of chloroform is added to the sample, centrifuge at 1500 rpm for 15 minutes and add the resulting supernatant to the wells with a prepared nutrient medium containing casein, 20 µl each, incubate at 35-37°C for 6-18 hours, proteolysis zones are shown by adding 5 ml of 5 to agar % aqueous solution of hydrochloric acid and evaluate the result by the size of proteolysis zones, while the size of the proteolysis zone of 5.3 ± 0.2 mm is regarded as a natural background of opportunistic microbiota, 8.37 ± 0.3 mm is a mild degree of periodontitis, 13, 3 ± 0.4 mm - moderate, 16.58 ± 0.2 mm - severe periodontitis.

Images