RU2279079C2 - Method for detecting protective activity microorganisms in fenton effect cases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves culturing microorganisms in liquid nutrient medium, separating supernatant from microbial cells by centrifuging. Control is concurrently prepared as liquid nutrient medium. Then, each sample is treated with chloroform and separating supernatant from chloroform by centrifuging. Next to it, Staphylococcus aureus test culture is added to case and control samples, incubated, test culture cells are separated by centrifuging and microbial suspension is prepared from them. Hydrogen peroxide solution is added to each sample and they are incubated. The test culture cells are separated by centrifuging, nutrient medium is added and incubated. Optical density of grown-up cultures is measured in case and control samples and test culture biomass live weight gain in case sample is determined from formula P=[(OD^CS - OD^C ]/ OD^C] *100%, where P is the biomass liveweight gain in case sample compared to control; OD^C is the optical density of control test culture; OD^CS is the optical density of case test culture. Then, conclusions are drawn if protective activity microorganisms are available in Fenton effect by interpreting test culture biomass live weight gain data in case sample.

EFFECT: wide range of functional applications in studying microbial cell physiology in medium containing small quantities of hydrogen peroxide.

1 dwg, 2 tbl

Description

The invention relates to microbiology and can be used in the research work of the research institute and in the practical work of bacteriological laboratories of clinical institutions that study the properties of microorganisms that characterize intermicrobial interactions and play a role in the formation of normal microbiocenoses of the human body.

Known generation in biological systems of reactive oxygen species, in particular the hydroxyl radical * OH (Free radicals in biology / Edited by W. Pryor. M: Mir, 1979. V.1. 318 S.). Its appearance is associated with reactions involving hydrogen peroxide and reduced metal ions of a transition valency, for example, iron (II) (Fenton reaction) (Ryan TP, Aust SD The role of iron in oxygen-mediated toxicities // Crit. Rev. Toxicol. - 1992. - Vol.22. P.119-141):

Н ₂ О ₂ + Fe ⁺² = * OH + OH ^- + Fe ⁺³

It is in the presence of small concentrations of hydrogen peroxide in the environment that a hydroxyl radical (* OH) is formed, which causes the death of microorganism cells as a result of the so-called Fenton effect (Sgibnev A.V. Regulation of the activity of bacterial catalase in intermicrobial interactions // Diss .... Cand. Biol Science. - Orenburg. 2002. - 150 p.) by oxidative damage to DNA (Smirnova GV, Muzyka NG, Glukhovchenko MN, Oktyabrsky ON. Hydrogen peroxide modulates intracellular levels of thiols and potassium in cells of Escherichia coli // Microbiology. 1998. T. 67. No. 5. S. 594-600. )

Microbiocenoses of the human body contain microorganisms that, by producing hydrogen peroxide, thereby prevent allochthonous microorganisms from populating the biotope (Hillier SL, Krohn MA, Klebanoff SJ, Eschenbach DA The relationship of hydrogen peroxide-producing lactobacilli to bacterial vaginosis and genital microflora in pregnant women / / Obstet. Gynekol. - 1992. - Vol. 79, No. 3. - P.369-373). At the same time, autochthonous microorganisms in microbiocenoses are quite resistant to the effects of hydrogen peroxide produced by microbes. Their resistance to high concentrations of hydrogen peroxide is explained by the stimulation of catalase activity (Menendez MC, Ainsa JA, Martin C., Garcia MJ katGI and katGII encode two different catalases-peroxidases in Mycobacterium fortuitum./ J. Bacteriol. 1997. Vol.179. (22. ) .P.6880-6886), whereas the resistance of microorganisms to the hydroxyl radical is not associated with catalase activity (McCormick ML, Buettner GR, Britigan BE Endogenous superoxide dismutase levels regulate iron-dependent hydroxyl radical formation in Escherichia coli exposed to hydrogen peroxide / / J. Bacteriol. 1998 V.180 (3), P.622-625) and may be determined by interbacterial interactions.

The objective of the proposed technical solution is to create a method for identifying a protective effect in microorganisms in the Fenton effect.

To solve this problem, in the inventive method for detecting the protective effect of microorganisms in the Fenton effect, the studied microorganism culture is grown in a liquid nutrient medium, the supernatant is separated from microbial cells by centrifugation, a control representing a liquid nutrient medium is prepared in parallel, then each of the samples is treated with chloroform and the supernatant is separated from chloroform by centrifugation, then a test culture of Staphylococcus aureus GISK No.201108 is added to the test sample and control, incubated, glue is separated ki test cultures by centrifugation and prepare microbial suspension from them, add hydrogen peroxide solution to each of the samples and incubate, separate the test culture cells by centrifugation, add nutrient medium to them, incubate, measure the optical density of the grown cultures in the experimental and control samples, determine the degree of biomass growth of the test culture in the experiment according to the formula

P = [(OD ^op -OD ^to ] / OD ^to ] · 100%,

where P is the degree of growth of the biomass of the test culture in the experiment compared with the control;

OD ^to - the optical density of the test culture in the control;

OD ^op - the optical density of the test culture in the experiment,

and then they judge the presence or absence of the protective culture of the studied culture of microorganisms in the Fenton effect by the degree of increase in the biomass of the test culture in the experiment.

Analogues of the invention in the patent and scientific literature are not found.

The authors experimentally established a new property of microorganisms - the ability to protect microorganisms from the oxidative effect of the Fenton effect, which occurs when small concentrations of hydrogen peroxide are present in the medium.

When studying the effect of supernatants on the survival of microorganisms under the influence of low concentrations of hydrogen peroxide, the authors discovered the ability of various types of microorganisms to protect other microorganisms populating this biotope from the oxidative effect of the Fenton effect. As a result of these studies, the assumption was made that microorganisms have substances that can protect microorganisms from the oxidative action of reactive oxygen species, in particular the hydroxyl radical formed as a result of the interaction of hydrogen peroxide with iron (II) ions. This assumption was confirmed by studies.

At the first stage of the study, the concentrations of hydrogen peroxide causing the Fenton effect were determined. For this, suspensions were prepared from museum cultures of Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis CCM 2124 (method for preparing suspensions: a 24-hour agar culture of microorganisms was washed with a sterile 0.9% sodium chloride solution and microbial suspensions were prepared whose optical density corresponded to 0.27 at 591 nm on SF-46). To 0.4 ml of the obtained microbial suspensions, 0.8 ml of hydrogen peroxide solution was added in concentrations from 0.5 to 60 mM with an interval of 5 mM, incubated for 30 minutes at 37 ° C. The incubated mixtures were centrifuged (3000 g for 15 minutes), the separated cells of microorganism cultures were washed twice with a sterile 0.9% sodium chloride solution and microbial suspensions were prepared from them, the optical density of which corresponded to 0.27 at 591 nm on SF-46. To 0.4 ml of the obtained microbial suspensions, 2.6 ml of liquid nutrient medium (meat-peptone broth) was added and incubated for 3-5 hours at 37 ° C with periodic stirring, then the optical density of the broth cultures was measured in the experiment and control at 591 nm on SF- 46. The research results are presented in the drawing, reflecting the survival of cultures of Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis CCM 2124 depending on the concentration of hydrogen peroxide.

As can be seen from the data presented in the drawing, the concentration of hydrogen peroxide, causing the Fenton effect, are 0.5-20 mm.

In the next stage of the study, cultures of Corynebacterium minutissimum, C. aquaticum, Staphylococcus epidermidis, S. aureus, Lactobacillus sp. Isolated from the female reproductive tract, and museum strains of Staphylococcus epidermidis CCM 2124 and S. aureus ATCC 6538P were grown in liquid nutrient medium (for each microorganism species used the appropriate type of nutrient medium) at 37 ° C for 24 hours. Microorganism broth cultures were centrifuged (3000 g for 15 minutes), the supernatant was separated from bacterial cells. Supernatants of various microorganisms were treated with chloroform for one hour (0.2 ml of chloroform per 2 ml of supernatant). At the same time, control samples were prepared — liquid culture media (for each sample the corresponding type of culture medium) was treated with chloroform for one hour (0.2 ml of chloroform per 2 ml of medium). Control and experimental samples were centrifuged to remove chloroform (3000 g for 15 minutes). 0.8 ml of prepared supernatants were poured into experimental tubes, 0.8 ml of a liquid nutrient medium treated with chloroform was added to control tubes. 0.4 ml microbial suspension of cultures of microorganisms Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis CCM 2124 was added to the experimental and control tubes (suspension preparation method: the 24-hour agar culture of microorganisms was washed with a sterile 0.9% sodium chloride solution and microbial suspension was prepared the density of which corresponded to 0.27 at 591 nm on SF-46). The resulting mixture was incubated for 40-60 minutes at 37 ° C. The incubated mixtures were centrifuged (3000 g for 15 minutes), the separated cells of microorganism cultures were washed twice with a sterile 0.9% sodium chloride solution and microbial suspensions were prepared from them, the optical density of which corresponded to 0.27 at 591 nm on SF-46. To 0.4 ml of the obtained microbial suspensions, 0.8 ml of hydrogen peroxide solution was added in concentrations from 0.5 to 20 mM with an interval of 5 mM, incubated for 30 minutes at 37 ° C. The incubated mixtures were centrifuged (3000 g for 15 minutes), the separated cells of microorganism cultures were washed twice with a sterile 0.9% sodium chloride solution and microbial suspensions were prepared from them, the optical density of which corresponded to 0.27 at 591 nm on SF-46. 2.6 ml of a liquid nutrient medium (meat-peptone broth) was added to 0.4 ml of the obtained microbial suspensions and incubated for 3-5 hours at 37 ° C with periodic stirring, then the optical density of the grown cultures of experimental and control samples was measured at 591 nm per SF-46 and determined the increase in biomass of test cultures in the experiment compared with the control. The research results are shown in tables 1 and 2.

As can be seen from the tables, the supernatants of various types of microorganisms influenced the survival of Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis CCM 2124 under the conditions of the Fenton effect, namely, they increased it, based on which the authors concluded that the studied cultures of microorganisms under the Fenton effect were protective. The greatest protective effect of the studied cultures of microorganisms in relation to the test strains Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis CCM 2124 from the Fenton effect was recorded in a culture medium with a hydrogen peroxide concentration of 10 mM.

The authors conducted studies on the choice of indicator culture from Staphylococcus aureus ATCC 6538P and Staphylococcus epidermidis CCM 2124, which allows testing the presence or absence of the Fenton effect in the studied microorganisms.

The authors chose the Staphylococcus aureus ATCC 6538P strain as a test culture because of its greater sensitivity to the damaging effect of the Fenton effect and its greater resistance to high concentrations of hydrogen peroxide compared to Staphylococcus epidermidis (see drawing). The strain is deposited in the GISK them. L.A. Tarasevich under registration number 201108 (Catalog of strains of the All-Union Museum of Pathogenic Bacteria, issue 1. - M., 1982).

The proposed strain of Staphylococcus aureus ATCC 6538P grows on simple nutrient media in the form of yellow opaque colonies with a diameter of 2-3 mm, gram-positive, non-spore-forming, microaerophile, catalase-positive, grows on solid nutrient media with 10-15% sodium chloride, forms acid from sucrose, maltose, D-mannitol, D-mannose, D-trehalose, α-lactose, D-galactose, β-D-fructose, D-ribose, α-hemolytic, coagulase-positive, reduces nitrates, produces hyaluronidase alkaline phosphatase, arginine hydrolase, urease, fibrinolysis β-glycosis oxidase, deoxyribonuclease, heat-resistant nuclease, clumping factor.

The method is as follows:

1. The studied cultures of microorganisms are grown in 2-3 ml of liquid nutrient medium (for each type of microorganism, the corresponding type of nutrient medium is used) at 37 ° C for 24 hours.

2. Bouillon cultures are centrifuged (3000 g for 15 minutes), supernatants are separated from microbial cells.

3. Supernatants are treated with chloroform for one hour (0.2 ml of chloroform per 2 ml of sample).

4. At the same time, a control sample is prepared - a liquid nutrient medium (for each type of microorganism the corresponding type of nutrient medium) is treated with chloroform for one hour (0.2 ml of chloroform per 2 ml of sample).

5. Each of the samples is separated by centrifugation from chlorofort (3000 g for 15 minutes).

6. 0.8 ml of supernatants are poured into the test tube, 0.8 ml of the liquid nutrient medium treated with chloroform in the control tube.

7. Add 0.4 ml of S. aureus suspension to the experimental and control samples (suspension preparation method: wash the S. aureus 24-hour agar culture with a 0.9% sodium chloride solution and prepare a microbial suspension with an optical density of 0.27 at 591 nm on SF-46).

8. Incubate the experimental and control samples for 40-60 minutes at 37 ° C.

9. After incubation, the experimental and control samples are centrifuged (3000g for 15 minutes), the separated cells of the S. aureus test culture are washed twice with 0.9% sodium chloride solution by centrifugation (3000g for 15 minutes), and a microbial suspension is prepared, the optical density of which corresponds to 0.27 at 591 nm on SF-46.

10. In each of the samples with a volume of 0.4 ml add 0.8 ml of a solution of hydrogen peroxide at a concentration of 10 mm and incubated for 30 minutes at 37 ° C.

11. After incubation, the experimental and control samples are centrifuged (3000g for 15 minutes), the separated cells of the test culture are washed twice with a sterile 0.9% sodium chloride solution and microbial suspensions are prepared from them, the optical density of which corresponded to 0.27 at 591 nm per SF-46.

12. To each of the samples with a volume of 0.4 ml add 2.6 ml of meat-peptone broth and incubate for 3-5 hours at 37 ° C and periodic stirring, then measure the optical density of the grown cultures of microorganisms in the experimental and control samples at 591 nm per SF-46.

13. Determine the degree of growth of the biomass of the test culture in the experiment by the formula:

P = [(OD ^op -OD ^to ] / OD ^to ] · 100%,

where P is the degree of growth of the test culture in the experiment compared with the control;

OD ^to - the optical density of the test culture in the control;

OD ^op is the optical density of the test culture in the experiment and then the presence or absence of the studied culture of microorganisms with protective action in the Fenton effect is judged by the degree of biomass increase of the test culture in the experiment.

Examples of specific performance of the method

Example 1. A representative of the normoflora Corynebacterium minutissimum was isolated from the vagina of a healthy woman. The studied culture was grown in a liquid nutrient medium (meat and peptone broth) for 24 hours at 37 ° C, the supernatant was separated, treated with chloroform for one hour (0.2 ml of chloroform per 2 ml of supernatant). At the same time, a control sample was prepared — a liquid nutrient medium (meat-peptone broth), treated with chloroform for one hour (0.2 ml of chloroform in 2 ml of medium). The control and experimental samples were centrifuged to remove chloroform (3000 g for 15 minutes). 0.8 ml of prepared supernatants were poured into experimental tubes, 0.8 ml of a liquid nutrient medium treated with chloroform was added to control tubes. 0.4 ml microbial suspension of a culture of the microorganism Staphylococcus aureus ATCC 6538 P was added to the experimental and control tubes (suspension preparation method: the 24-hour agar culture of the microorganism was washed with a sterile 0.9% sodium chloride solution and a microbial suspension with an optical density of 0 was prepared , 27 at 591 nm on SF-46). The test and control samples were incubated for one hour at 37 ° C. The incubated mixtures were centrifuged (3000 g for 15 minutes), the separated cells of the test culture were washed twice with a sterile 0.9% sodium chloride solution and microbial suspensions were prepared from them, the optical density of which corresponded to 0.27 at 591 nm on SF-46. To 0.4 ml of the obtained microbial suspensions, 0.8 ml of a solution of hydrogen peroxide at a concentration of 10 mM were added, incubated for 30 minutes at 37 ° C. The incubated mixtures were centrifuged (3000 g for 15 minutes), the separated cells of microorganism cultures were washed twice with a sterile 0.9% sodium chloride solution and microbial suspensions were prepared from them, the optical density of which corresponded to 0.27 at 591 nm on SF-46. 2.6 ml of meat-peptone broth was added to 0.4 ml of the obtained microbial suspensions and incubated for 5 hours at 37 ° C with periodic stirring, then the optical density of the grown cultures was measured in the experiment and control at 591 nm on SF-46 and the presence of or the lack of the ability of the studied culture of microorganisms to protect microorganisms from the action of the Fenton effect according to the formula:

P (C. minutissimum) = [(0.179-0.145) / 0.145] · 100% = 20.1%,

where P (C. minutissimum) is the growth rate of the test culture in the experiment with Corynebacterium minutissimum;

0,145 - optical density of the test culture in the control;

0.179 is the optical density of the test culture in the experiment with C. minutissimum.

It was revealed that the supernatant of C. minutissimum increased the survival of the test strain under the conditions of the Fenton effect, on the basis of which it was concluded that the studied culture was able to protect the test strain from reactive oxygen species.

Example 2. A representative of Staphylococcus epidermidis microflora was isolated from the skin of a healthy man. To identify the protective effect microorganism in the isolated culture with the Fenton effect, studies were carried out according to Example 1. The degree of growth of the test culture was 0% (optical density of the test culture in the control was 0.145, in the experiment 0.145).

Based on the results obtained, it is concluded that the studied culture does not have a protective effect in the Fenton effect.

Thus, the use of the proposed method allows us to identify a new property of microorganisms - a protective effect in the Fenton effect that occurs when there are low concentrations of hydrogen peroxide in the medium, which can be used to study the physiology of microbial cells and to decipher the mechanisms of interaction of microorganisms in biocenoses.

Table 1 Survival of S. epidermidis CCM 2124 The concentration of H ₂ O ₂ , mm Control, optical density, conv. units C.minutissimum, optical density, conv. units % of control C.aquaticum, optical density, conv. units % of control S.epidermidis, optical density, conv. units % of control S.aureus, optical density, conv. units % of control Lactobacillus sp., Optical density, conv. units % of control S.epidermidis CCM 2124, optical density, conv. units % of control S.aureus ATCC 6538P, optical density, conv. units % of control 0 0.15 0.15 0 0.15 0 0.15 0 0.15 0 0.15 0 0.15 0 0.15 0 5 0.131 0.142 8.40 0.134 2.29 0.132 0.76 0.131 0 0.138 5.34 0.132 0.76 0.131 0 10 0.116 0.138 18.97 0.119 2.59 0.118 1.72 0.118 1.72 0,126 8.62 0.118 1.72 0.117 0.86 fifteen 0.132 0.143 8.33 0.135 2.27 0.134 1,52 0.132 0 0.139 5.30 0.133 0.76 0.132 0 twenty 0.14 0.144 2.86 0.142 1.43 0.141 0.71 0.14 0 0.14 0 0.14 0 0.14 0 Protective effect Yes Yes Yes Yes Yes Yes Yes

table 2 Survival of S. aureus ATCC 6538P The concentration of H ₂ O ₂ , mm Control, optical density, conv. units C.minutissimum, optical density, conv. units % of control C.aquaticum, optical density, conv. units % of control S.epidermidis, optical density, conv. units % of control S.aureus, optical density, conv. units % of control Lactobacillus sp., Optical density, conv. units % of control S.epidermidis CCM 2124, optical density, conv. units % of control S.aureus ATCC 6538P, optical density, conv. units % of control 0 0.19 0.19 0 0.19 0 0.19 0 0.19 0 0.19 0 0.19 0 0.19 0 5 0.159 0.179 12.58 0.165 3.77 0.164 3.14 0.16 0.63 0.162 1.89 0.162 1.89 0.16 0.63 10 0.145 0.174 20.00 0.151 4.14 0.15 3.45 0.146 0.69 0.148 2.07 0.147 1.38 0.146 0.69 fifteen 0.163 0.181 11.04 0.169 3.68 0.166 1.84 0.164 0.61 0.166 1.84 0.165 1.23 0.164 0.61 twenty 0.181 0.186 2.76 0.183 1.10 0.186 2.76 0.182 0.55 0.184 1.66 0.184 1.66 0.182 0.55 Protective effect Yes Yes Yes Yes Yes Yes Yes

Claims (1)

A method for detecting a protective effect in microorganisms in the Fenton effect, namely, that the studied culture of microorganisms is grown in a liquid nutrient medium, the supernatant is separated from microbial cells by centrifugation, a control representing a liquid nutrient medium is prepared in parallel, then each of the samples is treated with chloroform and the supernatant is separated from chloroform by centrifugation, then a test culture of Staphylococcus aureus GISK No. 201108 is added to the test sample and control, incubated, the cells of the test culture are separated by centrifugation and microbial suspension is prepared from them, a hydrogen peroxide solution is added to each of the samples and incubated, the test culture cells are separated by centrifugation, nutrient medium is added to them, incubated, the absorbance of the grown cultures in the experimental and control samples is measured, the degree of biomass growth is determined. -cultures in the experiment according to the formula P = [(OD ^op -OD ^to ] / OD ^to ] • 100%, where P is the degree of growth of the biomass of the test culture in the experiment compared with the control; OD ^to - the optical density of the test culture in the control; OD ^op - the optical density of the test culture in the experiment, and then they judge the presence or absence of the protective culture of the studied culture of microorganisms in the Fenton effect by the degree of increase in the biomass of the test culture in the experiment.