RU2235324C1 - Method for assay of caseinolytic activity of microorganisms in express-diagnosis of intestine dysbacteriosis - Google Patents

Abstract

FIELD: medicine, biochemistry.

SUBSTANCE: assay method is carried out in microorganism-free supernatant feces fractions suing chloroform. Supernatant in the amount 20 mcl is added into well with special nutrient medium where casein is added also. Casein used as 1.5% solution prepared with 0.2 N NaOH is added to melted nutrient agar in the ratio 1;10, autoclaved at 121 C for 15 min, pH value is brought about to 7.2-7.4 and poured in sterile Petri dishes. After cooling wells with diameter 6 mm are made in agar in dishes. After culturing in thermostat at 37 C for 6-8 h proteolysis zones are developed by addition of 5 ml 5% aqueous solution of hydrochloric acid on agar. Diameters of proteolysis zones are measured in millimeters (mm). After measurement of proteolysis zone diameters activity is considered to be low if zone diameters is below 10 mm, mean - at 11-16 mm and high - at above 16 mm of proteolysis diameter zones. Invention provides rapid and easy assay.

EFFECT: improved assay method.

Description

The invention relates to medicine, namely to gastroenterology, microbiology and biochemistry. Recently, with a wide variety of external influences (environmental pollution, increased radiation background, magnetic disturbances, etc.), extreme conditions, stressful situations, physical inactivity, biorhythm disturbances, in cases of decreased immunological status, development of pathological conditions in the gastrointestinal tract and of inflammatory processes of both infectious and non-infectious etiology, environmental shifts occur in the microbial landscape of the intestine (Sokolova K.Ya., Solovieva IV Dysbacteriosis. Theory and practice . Ka N. Novgorod, 1999; Efimov BA, Volodin NN Kafarskaya LI et al Characterization of microorganisms, colonizing the human gut mikrobiol Journal 2002, 5: 98-104).....

The development of intestinal dysbiosis of various etiologies aggravates the course of the underlying disease, worsens its prognosis. In some cases, dysbacterioses subsequently become a determining factor in the formation of the pathological state of the body, lead to pronounced functional disorders in the digestive tract and can cause the development of an infectious disease. According to many authors, intestinal dysbiosis is observed among 70-90% of patients with gastrointestinal, cardiovascular, neurological, allergic and other diseases (Vorobyov A.A., Abramov N.A., Bondarenko V.M., Shenderov B .A. Dysbacteriosis - an urgent problem of medicine.Vest. RAMS, 1997, 3: 4-7. Salminen S., Isolauri E., Onnela T. Gut flora in normal and disordered states. Chemotherapy. 1995, 41 (suppl.l) : 5-15).

In medical practice, for the diagnosis of intestinal dysbiosis, as a rule, a classic bacteriological analysis is used, which requires a large number of expensive nutrient media and takes a long time (at least 5 days). It should be noted that about 500 species of microorganisms of normal microflora live in the human intestine alone. At the same time, the ratio of anaerobes to aerobes is 1000: 1, and it is practically impossible to take into account all microorganisms that are so distant in a taxonomic relation with classical bacteriological analysis.

For screening studies for dysbiosis, the development of sufficiently fast biochemical methods that are easily implemented in practice is required.

An analog, according to the authors, is the 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).

Principle. In the interaction of bacterial culture proteinases with casein contained in an agar medium, its cleavage occurs. When a protein coagulant is added against a cloudy agar background, zones of enlightenment around the growth of caseinolytically active microorganisms are detected.

Reagents and materials. The studied daily culture of microbes on the BCH or MPA; a solution of 1% casein in 1/15 M phosphate buffer pH 7.2; 2% agar in saline, pH 7.2, in vitro or cone; developers for protein (casein): a) a 10% solution of trichloroacetic acid in a bottle 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) - amid black 10 V or kumashi bright blue R-250 in dark glass bottles with ground glass stopper; test tubes, pipettes, Petri dishes, slides, punches for creating holes (wells) in agar and other laboratory equipment.

Preparation of the dye solution used to color casein protein precipitates: amid black 10 V (or kumashi bright blue R-250) 50.0 g + ethanol 96 ° 4.5 l + glacial acetic acid 1.0 l + dist. water 4,5 l. The paint is dissolved and the solution is left overnight at room temperature, then filtered and used. The use of dye increases the sensitivity of the method.

The method of making casein in agar. In 10 ml of molten and cooled to 60-65 ° С 2% agar, 1 ml of 1% casein solution is poured in a test tube, mixed and the contents are quickly poured into a sterile Petri dish or on 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 (slides) with crops are placed in a thermostat at 37 ° C. After 6-12 hours, the developer “a” or “b” is applied to the surface of the agar with samples in a volume of 4-5 ml (the samples on a glass slide are lowered into a cup with the developer), incubated for 7-10 minutes and removed.

Registration of results is carried out visually. Against the turbid background of casein agar with a positive result, zones of enlightenment with caseinolytic microbial cultures are noted. In a negative reaction, the agar medium around the well remains uniformly cloudy. The use of dyes helps to increase the sensitivity of the method, since turbid casein precipitates become more contrasting, becoming black and blue, and lysis zones, on the contrary, are unpainted and bright and are better detected, especially weak degrees of bacterial caseinolysis.

Some of the disadvantages of the proposed method are:

- poor solubility of casein in phosphate buffer,

- uneven its introduction into the nutrient medium.

As a prototype, the authors propose a biochemical rapid method developed in the laboratory of bacterial virulence genetics of the NIIEM them. N.F. Gamalei (Luchshev L.I., Bondarenko V.M., Isaeva N.P., Zemskova L.I., Shakhmardanov M.Z., Gritsevskaya N.Yu. Indirect method for rapid diagnosis of intestinal dysbiosis 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 an approximate response after 18 hours from the start of the study.

Caseinolytic activity testing was performed on Petri dishes with casein agar after an 18-hour incubation in an incubator. Zones of proteolysis showed the addition of 5% trichloroacetic acid.

Activity was considered low with zone diameters up to 10 mm, medium at 11-15 mm and high with 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 a nutrient medium for determining caseinolytic activity.

The authors propose a method for determining the caseinolytic (proteolytic) activity of microorganisms in the rapid diagnosis of dysbiosis in feces supernatants.

For analysis, portions of feces were collected in a sterile dish or the material was collected using a sterile probe with a dekron or cotton swab, which was inserted into the rectum as high as possible, and by rotating movements, avoiding contact with the intestinal walls, material was collected, which was transferred to a test tube with physiological saline. Before sending to the laboratory, samples can be stored for several days at + 5-8 ° C.

The study of the proteolytic (caseinolytic) activity of feces supernatants was carried out in a portion of feces 1-2 g, which was diluted with saline 1:10. After 20 minutes of settling, the liquid above the precipitate was aspirated and transferred to test tubes, not more than 0.05 ml (1 drop) of chloroform (for lysis of bacteria and disinfection) was added to the sample, sedimented for 30-40 minutes and introduced into wells (no more than 9 per cup) with a specially prepared nutrient medium of 20 μl. A caseinolytically active sample with a known degree of proteolysis was introduced into one of the control wells.

A method of preparing a nutrient medium with casein consists of the following steps. First, 100 ml of a 3.5% standard nutrient agar containing yeast or fish extract and other ingredients (manufactured in Makhachkala and others) for 5 Petri dishes was prepared and brought to a boil. Then prepared a 1.5% solution of casein on 0.2 N NaOH (0.15 g of casein per 10 ml of alkali). In this case, complete dissolution of casein in an alkaline solution was achieved. After that, nutrient agar and casein solution were mixed 10: 1, autoclaved at 121 ° C for 15 min, adjusted to pH 7.2–7.4 and poured into sterile Petri dishes. After cooling the cups on a flat surface in the agar, holes with a diameter of 6 mm were made with a special punch or stamp. Thus, the nutrient medium with casein was ready for introducing fecal supernatants into the wells to determine caseinolytic (proteinolytic) activity in them.

After cultivation in a thermostat at 37 ° C for 6-8 h, the proteolysis zones were manifested by adding 5 ml of a 5% aqueous hydrochloric acid solution to agar. In this case, it is not necessary to additionally tint the agar for the manifestation of 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 its use in screening studies.

The proposed method allows you to completely dissolve casein and make it without residue in a nutrient medium. In this case, you can increase the concentration of casein in the solution, which increases the sensitivity of the determination. The cultivation time in the thermostat is reduced to 6-8 hours. When the reaction manifests itself, additional coloring of the medium is not required, i.e. the additional reaction stage is excluded, which makes the method more economical in time and in terms of material costs.

The diameters of the proteolysis zones were measured in millimeters (mm). As a result of measuring the proteolysis zones on our medium, activity was considered low when the diameter of the zones is up to 10 mm, medium - at 11-16 mm, high - with the diameter of the proteolysis zones above 16 mm.

A parallel study of feces for dysbiosis using classical bacteriological analysis and using the express method performed on a large number of patients (543 people) showed a correlation between a decrease in the population level of anaerobic obligate and optional normal microflora, and an increase in the number of aerobic conditionally pathogenic gram-negative microflora and the size of the proteolysis zones of feces supernatants. This is due to an increase in the number of bacteria producing various proteases.

The coincidence of the results when using the classical and express methods was 89.8%. There was a direct correlation between the diameters of the proteolysis zones and the severity of dysbiosis, which can be used for diagnostic purposes and serve as a criterion for the effectiveness of the therapy.

Claims (1)

A method for determining the caseinolytic activity of microorganisms in the rapid diagnosis of intestinal dysbacteriosis, including determining the proteolytic activity of feces supernatants by lysis zones on a nutrient medium, characterized in that a portion of feces placed in a test tube weighing 1-2 g is diluted with saline solution 1:10, after 20 min settling liquid is sucked off and transferred to test tubes, no more than 0.05-0.1 ml of chloroform is added to the sample, after settling for 30-40 minutes, 20 μl of soup are added to wells with nutrient medium natant, and in one of the wells - a control caseinolytically active sample, after which it is cultivated in a thermostat for 6-8 hours and the lysis zones are shown by adding 5 ml of a 5% aqueous hydrochloric acid solution on agar and the diameters of the enlightenment zones are measured, and the medium is prepared from 100 ml of standard nutrient agar, brought to a boil, prepare a 1.5% casein solution on 0.2 N NaOH and bring it to complete dissolution in an alkaline solution, after which nutrient agar and casein solution are mixed 10: 1, autoclaved at 121 ° From 15 min, adjust the pH to 7.2-7.4 and dec vayut into sterile petri dishes and, after cooling make holes 6 mm in diameter in an amount of not more than 9.