WO2013100792A1

WO2013100792A1 - Method for accelerating bacterial biomass growth and attenuating the virulence of bacteria

Info

Publication number: WO2013100792A1
Application number: PCT/RU2011/001060
Authority: WO
Inventors: Артур Викторович МАРТЫНОВ; Борис Славинович ФАРБЕР; Софья Борисовна ФАРБЕР
Original assignee: Martynov Artur Viktorovich; Farber Boris Slavinovich; Farber Sof Ya Borisovna
Priority date: 2011-12-28
Filing date: 2011-12-28
Publication date: 2013-07-04
Also published as: EA201300205A1; EA025623B1; IN2014MN01483A

Abstract

The invention relates to biotechnology and medicine. The method for accelerating biomass growth and attenuating the virulence of bacteria involves using intracellular protein phosphorylation inducers, including the cyclic adenosine monophosphate accumulation activators papaverine, bendazole and dipyridamole in the form of salts or bases, as inhibitors of the virulence of microorganisms. A mixture of intracellular protein phosphorylation inducers is used as low molecular weight accelerators of microorganism growth. Papaverine, bendazole and dipyridamole in the form of salts or bases in specific concentrations are used as a cyclic adenosine monophosphate accumulation activator mixture. In order to attenuate the virulence of bacteria, the mixture is administered to a contagious patient parenterally, perorally, rectally or by application to the affected region 1-10 days prior to administration of an antibiotic or in parallel therewith.

Description

A method of accelerating the growth of bacterial biomass and suppressing the virulence of bacteria

Technical field

The invention relates to medicine, veterinary medicine and biotechnology, namely, the production of genetic engineering and other biotechnological products, and is intended for the treatment of infectious diseases of humans and animals, as well as in biotechnology to enhance the growth of the biomass of microorganisms and the production of medical and veterinary biologics, food additives, probiotics and lactic acid starter cultures, vaccine production.

State of the art

Recently, there has been an intensification of the development of biotechnological methods for obtaining the biomass of microorganisms for the production of protein-based preparations, a significant percentage of which belong to products of bacterial origin (vaccines, toxoids, probiotics, etc.) [1]. One of the ways to solve the problem of optimizing the synthesis of biotechnological protein and non-protein components and increasing microbial mass is the search and modeling of microorganism growth stimulators. Recently, many scientific papers have been published on the stimulating effect of various physical, chemical, and other factors on the biological properties of cells [2]. A wide spectrum of biological activity is characterized by stimulants of chemical origin - imidazole, isoquinoline and their derivatives, which are part of the structure of many natural and synthetic compounds that can induce the growth rate of the microbial population [1]. The use of enhancers is an important achievement in biotechnological productions; they can increase both the percentage of output of biotechnological protein products and the increase in microbial mass outside the physiological norm by an order of magnitude and higher. So there is a theoretical possibility significantly accelerate the rate of accumulation of microbial mass and the synthesis of protein products by microorganisms. Increased growth and enzymatic activity of microorganisms contribute to an increase in the biomass of cells and, accordingly, metabolites for their further effective application in various sectors of the economy, in particular, in the biotechnological, medical, and pharmaceutical industries.

In order for a pathogenic microorganism to cause an infectious disease, it must possess one characteristic - virulence - the ability not only to penetrate into the macroorganism, multiply in it, but also to suppress its protective mechanisms, which results in the development of an infectious disease. Virulence is a sign not of a species, like pathogenicity, but of strain, i.e. inherent not to the whole species, but to specific strains. Virulence can also be defined as the phenotypic manifestation of the pathogenic genotype of microorganisms. As a quantitative sign of virulence, in contrast to a qualitative one - pathogenicity, it has units of measurement. It is measured by the quantity, i.e., the dose of microorganisms that cause a certain biological effect. It can be:

- DCL (dosis certae letalis) is an absolutely lethal dose - the minimum amount of the pathogen that causes the death of 100% of laboratory animals taken in the experiment;

- DLM (dosis letalis minima) is the minimum lethal dose - the minimum amount of the pathogen causing the death of 95% of laboratory animals taken in the experiment; - LD50 is the minimum amount of the pathogen that causes the death of 50% of laboratory animals taken in the experiment (used to measure virulence most often).

In this case, the type of laboratory animal at which the given dose was determined is always indicated, since the sensitivity of different types of laboratory animals to various microorganisms is different. The method of introducing a culture of microorganisms is also indicated necessarily - intraperitoneally, intramuscularly, intranasally, intravenously.

Virulence is a labile sign. It can vary both upward and downward, both in vivo and in vitro. With a maximum reduction in virulence, pathogenic microorganisms can become avirulent, that is, non-virulent, but virulent microorganisms are always pathogenic.

Virulence is realized through a series of sequential processes of interaction of microbial cells with cells and tissues of a macroorganism: adhesiveness - the ability to attach to cells;

colonization - the ability to reproduce on their surface;

invasiveness - the ability to penetrate into cells and adjacent tissues and the formation of biologically active products, including toxins.

Adhesion of microorganisms to receptors of sensitive cells of a macroorganism is an essential element of their interaction, since if microorganisms do not adhere, then usually they do not multiply, but are removed from the body. Many microorganisms in the process of evolution have acquired special morphological and chemical structures that provide adhesion. These include villi and adhesins - specific structures (proteins and carbohydrates) on the surface of a microbial cell, corresponding to the receptors of cells of a macroorganism.

The known method and composition, as well as methods for their use, which can significantly accelerate the growth of microorganisms in vitro. Microorganisms, for example, fungi or bacterial cells, when cultured on nutrient media in the presence of exogenous enhancers, such as picolinic acid and metal picolinates, grow much faster and in larger quantities. For example, chromium picolinate enhances the growth of yeast by 30 times (the number of colonies), with the average cell growth index in the presence of stimulants ranging from 0.4 to 0.5 times [ ² ]. With a significant number of colonies (a 30-fold increase in the number of colonies), the increase in the number of world organisms was still insignificant (15–20% more than in the control), in addition, salts of picolinic acid and heavy metals — chromium, zinc — were strong activators. and iron, which in biotechnological production is a negative factor due to the risk of interaction of the biotechnological product with heavy metals. This is important in connection with the mandatory testing of heavy metals biotechnological products. A method for suppressing virulence of bacteria by adding a culture medium of effective amounts of phenylpropanoid inhibitors is also known [ ³ ]. The disadvantage of this invention is the apparent genotoxicity of the added component and the impossibility of its use in medicine. Although the substance suppressed the expressed virulence factors, it only affected genes, and it was irreversible. Such bacteria, although they lost their virulence factors, became obvious mutants and inherited signs of low virulence in future generations. Disclosure of invention

The basis of the invention is the task of developing microbial growth activators that do not contain heavy metals, are non-toxic to humans and animals, do not have a mutagenic and carcinogenic effect and increase the growth of the number of microbial cells by 2 or more times, and also have the ability to suppress virulence microorganisms, which allows them to be used in the treatment of infectious diseases in humans and animals through the use of enhancers before a course of antimicrobial therapy.

The problem is solved by adding to the nutrient medium for the cultivation of microorganisms a mixture of microorganism growth activators with proven harmlessness to the human body, which leads to an acceleration of the appearance of the first colonies and an increase in the number of microbial cells by 2-4 times, as well as for the treatment of infectious diseases as inhibitors of virulence of microorganisms before or in parallel with a course of antimicrobial therapy. Moreover, activators of the accumulation of cyclic adenosine monophosphate, such as papaverine, bendazole and dipyridamole, where their mass ratio varies from 1: 100 to 100: 1 and in the range of concentrations in the nutrient medium from 0.0001 g /, are used as activators of the growth of microorganisms. % to 0.1 g /%., and papaverine, bendazole and dipyridamole can be both in the form of salts, and in the form of bases, and for therapeutic purposes, they can be administered orally, injection or rectally to a person before using the antibiotic or together with him.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1.- The use of growth enhancers in increasing the biomass of P.

aeruginosa for subsequent use in vaccine production

The dynamics of the accumulation of microbial mass is important for obtaining nutrients of microbial origin. Allosteric activators of cyclic adenosine monophosphate increase the metabolism of bacteria. As a result of such exposure, the number of microbial cells increases.

When enhancers were added at a concentration of 0.1 ± 0.05%, the number of microorganisms did not significantly differ from the number of microorganisms when cultured on a control medium higher. At a concentration of enhancers 0.01 ± 0.005% of the number of microbial cells increased. The maximum number of microorganisms was achieved with the addition of enhancer A at a concentration of 0.01 ± 0.005% (Table 3.8).

In addition, under the influence of enhancers, increased pigmentation was observed - the colonies became saturated green and visually increased in size.

As the data in table 1 show, the number of microbial cells under the influence of activators increases compared with the control (medium without enhancers).

Table 1.- The number of microbial cells of P. aeruginosa when cultured on nutrient agar with the addition of enhancers at a seed dose of 10 ⁶

Notes: N is the average value of the number of microbial cells of microorganism strains, billion / ml, n is the average deviation, V is the reproduction rate, cells / hour.,% - This is the mass / volume, A is papaverine hydrochloride, B is bendazole, C - dipyridamole ascorbate, * - the difference in indicators is statistically significant (p <0.05).

The number of microbial cells at 0.1 ± 0.05% of the concentration of enhancer A was (4.2 ± 0.2) - (5.1 ± 0.4) x 10 ⁹ / ml. The number of microbial cells at 0.1 ± 0.05% of the concentration of enhancer B was (4.3 ± 0.3) - (4.6 ± 0.5) x 10 ⁹ / ml. The number of mic of rodent cells at 0.1 ± 0.05% of the enhancer C concentration was (4.1 ± 0.2) - (5.8 ± 0.7) x 10 ⁹ / ml.

At 0.01 ± 0.005% concentration of enhancer A, the number of microbial cells increases to (5.0 - 5.5) x 10 ⁹ / ml. The number of microbial cells at 0.01 ± 0.005% of the concentration of enhancer B was (4.8 ± 0.5) - (5.4 ± 0.3) x 10 ⁹ / ml. The number of microbial cells at 0.01% concentration of enhancer C was (4.8 ± 6.3) - (6.1 ± 0.7) × ^{10 9} / ml. The concentration of 0.001% enhancer A promotes the accumulation of microbial cells up to (6.0 - 6.5) x 10 ⁹ / ml. The number of microbial cells at 0.001% enhancer B was (5.2 ± 0.2) - (5.6 ± 0.5) x 10 ⁹ / ml. At 0.001 ± 0.0005% concentration of enhancer C, the number of microbial cells was (3.2 ± 0.2) - (5.2 ± 0.2) x 10 ⁹ / ml.

Thus, both in the formation of colonies and in the accumulation of microbial cells, the activity of enhancer A is 0.001 ± 0.0005%, which contributes to the accumulation of microbial cells by a factor of 5–2 compared with other activators growth. That is, the formation of the largest number of microbial cells on nutrient agar was observed at an enhancer concentration of 0.001 ± 0.0005% A and amounted to (5.8 ± 0.4) - (6.3 ± 0.6) billion / ml.

While the smallest number of microbial cells was observed at an enhancer concentration of 0.1 ± 0.05% B and amounted to (4.3 ± 0.4) - (4.6 ± 0.5) billion / ml.

The results of statistical data processing table. 1. indicate that the differences between the number of microbial cells when using enhancers of different concentrations are statistically significant, which indicates the effectiveness of using enhancers to increase the number of microbial cells.

Clinical strains, due to their special adaptation to adverse conditions, form resistance to many factors, in particular drugs. Therefore P.aeruginosa strains were selected for antibiotic resistance with a polyvalent to test the effects of growth stimud1yatoro ^{^} Bend (Table. 2). Table 2 .- The number of P. aeruginosa microbial cells (clinical strains) when cultured on nutrient agar with the addition of enhancers at a seed dose of 10 ⁶

Notes: N is the average value of the number of microbial cells of the microorganism, billion / ml, p is the average deviation, V is the rate of reproduction, cells / hour.,% Is mass / volume, A is papaverine hydrochloride, B is bendazole, C is dipyridamole ascorbate, * - the difference in indicators is statistically significant (p <0.05).

As the data in Table 2 show, the number of P. aeruginosa microbial cells (clinical strains) increases under the influence of activators compared to control (medium without enhancers). Thus, when grown on nutrient agar, the number of microbial cells of the clinical P. aeraginosa strains Ν ° 25 and M ° 53 significantly increases in comparison with the P. aeraginosa M ° 15 strain.

The greatest number of microbial cells of clinical P. aeraginosa strains is formed on nutrient agar with the addition of enhancers at a concentration of 0.0001 ± 0.00005% A and amounted to (4.8 ± 0.4) - (7.6 ± 0.6) billion. / ml

Whereas the smallest number of microbial cells was with the addition of enhancers at a concentration of 0.1% A and amounted to (3.5 ± 0.1) - (3.8 ± 0.4) billion / ml.

The results of the statistical processing of the data in Table 2 show that the differences between the number of microbial cells when using enhancers A, B or C in a concentration of from 0.001 ± 0.0005% to 0.1 ± 0.05% and control are statistically significant. This indicates the effectiveness of the use of growth stimulants of various concentrations to increase the number of microbial cells.

Next, we checked how the number of microbial cells changes during the cultivation of P. aeraginosa on media obtained from erythrocyte mass with the addition of various concentrations of enhancers (Table 3).

Table 3 .- The number of microbial cells of P. aeraginosa when cultured on media with erythrocyte mass with different concentration of enhancers at a sowing dose of 10 ⁶

Culture medium - erythromass

Enhancers, Pseudomonas Pseudomonas Pseudomonas concentration aeraginosa aeraginosa aeraginosa

ATCC 27853 ATCC 9027 66-16

N ± n V N ± n V N ± n V

0.1 ± 0.05% A 2.2 ± 0.1 * 1.01 2.8 ± 0.4 * 1, 03 _2.9 ± 0.4 * _LQ3_

0.1 ± 0.05% V 2.3 ± 0.3 * 1.02 2.6 ± 0.5 * 1.02 2.4 ± 0.3 * 1.02

0.1 ± 0.005% C 3.8 ± 0.7 * 1, 05 2.5 ± 0.2 * 1.02 2.2 ± 0.2 * 1, 01

0.01 ± 0.005% A 4.3 ± 0.3 * 1.05 4.2 ± 0.4 * 1.05 4.7 ± 0.5 * 1.06

0.01 ± 0.005% B 4.8 ± 0.5 * 1.06 4.4 ± 0.2 * 1.0 05 4.3 ± 0.3 * 1.05

0.01 ± 0.005% C 4.1 ± 0.7 * 1.05 4.9 ± 0.4 * 1.06 4.8 ± 0.3 * 1.06

0.001 ± 0.0005% A 3.8 ± 0.4 * 1.05 4.1 ± 0.6 * 1.03 4.2 ± 0.4 * 1.04 0.001 ± 0.0005% B 4.0 ± 0.2 * 1.05 3.9 ± 0.5 * 1.04 3.9 ± 0.5 * 1.05

0.001 ± 0.0005% C 3.8 ± 0.2 * 1.03 4.0 ± 0.2 * 1.03 4.0 ± 0.4 * 1.05

Control 3.2 ± 0.2 1.04 3.4 ± 0.1 1.04 3.5 ± 0.3 1.04

Notes: N is the average value of the number of microbial cells of microorganisms, billion / ml, p is the average deviation, V is the reproduction rate, cells / hour.,% Is mass / volume, A is papaverine hydrochloride, B is bendazole, C is dipyridamole. ascorbate, * - The difference in indicators is statistically significant (p <0.05).

As can be seen from the data table. 3, the following indicators of the number of grown microbial cells were obtained when cultured on erythrocyte mass media with the addition of enhancers: at a concentration of 0.1 ± 0.05%, the number of microbial cells was (2.2-3.8) billion / ml independently from P. aeruginosa strain and enhancer species (A, B or C). At a concentration of 0.01 ± 0.005%, the number of microbial cells increases and amounts to (4.1–4.9) billion / ml for all P. aeruginosa strains and all types of enhancers.

At a concentration of 0.001 ± 0.0005%, the number of microbial cells of P. Aeruginosa decreases than at a concentration of 0.01 ± 0.005% and amounts to (3.3 - 4.0) billion / ml. That is, the greatest number of microbial cells is observed when enhancers are added at a concentration of 0.01%. In control, this indicator was (3.2 - 3.5) billion / ml.

That is, the largest number of microbial cells on media obtained with erythrocyte mass was observed at an enhancer concentration of 0.001 ± 0.0005% B and was equal to (3.6 ± 0.5) - (4.0 ± 0.2) billion . / ml, and the highest reproduction rate was 1.05 cells / hour. While the smallest number of microbial cells was observed at an enhancer concentration of 0.1 ± 0.05% B and was equal to (2.3 ± 0.3) - (2.6 ± 0.5) billion / ml, and the smallest the reproduction rate was 1, 02 cells / hour.

The results of statistical data processing table. 3 indicate that the differences between the number of microbial cells when using enhancers A, B or C in a concentration of from 0.001 ± 0.0005% to 0.1 ± 0.05% and control are statistically significant. This indicates the effectiveness of the use of growth stimulants A, B or C of various concentrations to increase the number of microbial cells.

How does the number of microbial cells change upon cultivation of P. aeruginosa on media obtained from grain stillage, with the addition of various concentrations of Tracer enhancers are presented in table. four.

Table 4 .- The number of P. aeraginosa microbial cells when cultured on media from grain stillage with the addition of various concentrations of enhancers at a seed dose of 10 ⁶

As can be seen from the data table. 4, the following indicators of the number of grown microbial cells were obtained upon cultivation on media from grain stillage with the addition of enhancers: at a concentration of 0.1 ± 0.05%, the number of microbial cells was (4.2-5.8) billion . / ml regardless of the strain P. aeraginosa and the type of enhancers (A, B or C). At a concentration of 0.01 ± 0.005%, the number of microbial cells increases and amounts to (6.2 - 6.8) billion / ml for all P. aeraginosa strains and all types of enhancers. At a concentration of 0.001 ± 0.0005%, the number of P. aeraginosa microbial cells is less than at a concentration of 0.01 ± 0.005% and amounts to (5.1-5.4) billion / ml. I.e, 11 001060

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the greatest number of microbial cells is observed when enhancers are added at a concentration of 0.01 ± 0.005% and amounts to (6.2-6.8) billion / ml. In the control, this indicator was equal to (3.2 - 3.5) billion / ml. Thus, on media obtained from grain stillage, the number of grown microbial cells using enhancers exceeds 2 times the number of cells that grow on control media, and is higher than the number of microbial cells on the erythrocyte mass.

That is, the largest number of microbial cells on media obtained from grain stillage was observed at an enhancer concentration of 0.01 ± 0.005% C and amounted to (6.4 ± 0.7) - (6.8 ± 0.4) billion / ml, and the highest reproduction rate was 1.08 cells. / hour

Whereas the lowest concentration of microbial cells was observed at an enhancer concentration of 0.1 ± 0.05% B and was equal to (4.3 ± 0.3) - (4.6 ± 0.5) billion / ml, and the varying rate of reproduction was 1.05 cells. / hour

The results of statistical data processing table. 4 indicate that the differences between the number of microbial cells when using enhancers A, B or C in a concentration of from 0.001 ± 0.0005% to 0.1 ± 0.05% and control are statistically significant. This indicates the effectiveness of the use of growth stimulants A, B or C of various concentrations to increase the number of microbial cells.

Further research was aimed at developing combinations of enhancers (Table 5).

Table 5.- The number of P. aeruginosa cells under the influence of a combination of enhancers at a seed dose of 10 ⁶

T U2011 / 001060

12

As the data in table. 5, the combination of two enhancers A and B at a concentration of 0.01 ± 0.005% increases the number of microbial cells by 3-4 times in comparison with the control and is (7.2-8.2) x 10 ⁹ / ml against (3 , 1-2, 8) x 10 ⁹ / ml in control. The combination of two enhancers A and C at a concentration of 0.01% increases the number of microbial cells by 4 times compared with the control and is equal to (8.3–9.4) x 10 ⁹ / ml versus (3.1–2.8 ) x 10 ⁹ / ml in the control. Approximately the same data are observed with a combination of enhancers B and C.

The combination of three growth stimulators GA7B, 1 C ^ concentration of 0.01% increases the number of microbial cells by almost 6 times and is (10.5-1 1.8) x 10 ⁹ / ml. During cultivation of P. aeruginosa, the number of microbial cells with the addition of two enhancers A and B at a concentration of 0.001 ± 0.0005% was (9.3-10.4) x 10 ⁹ / ml, with the combined use of two enhancers A and C at a concentration of 0.001 ± 0.0005% was (9.9-10.8) billion / ml, with the addition of enhancers B and C at a concentration of 0.001 ± 0.0005% was (9.1-9.3) billion / ml. 1060

13

Whereas with the addition of three stimulants A, B, and C at concentrations of 0.001%, the number of microbial cells was (11.6 -12.6) x 10 ⁹ billion / ml, which is 6.5 times more than in the control. And the highest reproduction rate was 1.12 cells / hour. Whereas the smallest number of microbial cells was observed with a combination of enhancers A and B at a concentration of 0.01%, and the lowest reproduction rate was 1.08 cells / hour.

Thus, upon cultivation of P. aeruginosa on media obtained from grain stillage with the addition of a combination of enhancers, the reproduction rate increases by 1.2 times compared to the control. That is, the number of microorganisms that are cultivated on media from grain stillage with the addition of a combination of enhancers significantly increases compared to the number of bacteria that are cultivated on media obtained from red blood cells with the addition of a combination of enhancers, which indicates the use of media with grain stillage for cultivation of P. aeruginosa.

The results of statistical data processing table. 5 indicate that the differences between the number of microbial cells when using a combination of enhancers A, B, C in a concentration of from 0.001 ± 0.0005% to 0.1 ± 0.05% and control are statistically significant. This indicates the effectiveness of the use of growth stimulants A, B, C in combination in concentrations from 0.001 ± 0.0005% to 0.1 ± 0.05% to increase the number of microbial cells. How the number of microbial cells changes during cultivation of P. aeruginosa under the influence of a combination of enhancers on erythrocyte mass media is shown in Table. 6.

Table B. The number of P. aeruginosa microbial cells under the influence of a combination

enhancers on erythrocyte mass media at a culture dose of 10 ⁶

Culture media - erythrocyte mass

Enhancers, Pseudomonas seudomonas Pseudomonas concentration aeruginosa aeruginosa aeruginosa

ATCC 27853 ATCC 9027 66-16

N ± n V N ± n V N ± n V

0.01 ± 0.005% A 4.5 ± 0.6 * 1.06 4.4 ± 0.8 * 1.05 4.3 ± 0.7 1.05 0.01 ± 0.005% B

0.01 ± 0.005% A 4.1 ± 0.9 * 1.05 4.9 ± 0.8 * 1.06 4.8 ± 0.7 * 1.06 11 001060

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As the data in table. 6, the combination of two enhancers A and B at a concentration of 0.01% increases the number of microbial cells compared to the control by a factor of 2 and is (4.3 - 4.5) x 10 ⁹ / ml versus (2.2 - 3, 1) x 10 ⁹ / ml in the control. The combination of two enhancers A and C increases the number of microbial cells to (4.1- 4.9) x 10 ⁹ / ml. The number of microbial cells also practically changes with the addition of a combination of two enhancers B and C. The combination of three growth stimulants A, B and C at a concentration of 0.01% increases the number of microbial cells by 3 times and amounts to (5.3 - 5, 8) billion / ml.

When P. aeruginosa is cultivated on red blood cell media, the number of cells with the addition of two enhancers A and B at a concentration of 0.001 ± 0.0005% is (4.5-4.7) x 10 ⁹ / ml, with a combination of two of enhancers A and C in the same concentration, the number of cells was (3, 8-4, 1) x 10 ⁹ / ml, with the addition of enhancers B and C at a concentration of 0.001%, the number of cells was (3.7-4.0) x 10 ⁹ / ml.

Whereas with the addition of three stimulants A, B, and C at a concentration of 0.001%, the number of cells was (6.2-6.4) x 10 ⁹ / ml, which is 3 times more than in the control. Thus, the highest reproduction rate was 1.08 cells / hour. with the addition of three stimulants A, B and C at a concentration of 0.001 ± 0.0005%.

Thus, the number of microorganisms cultivated on media obtained from erythrocyte mass with the addition of a combination of enhancers significantly increases, which indicates the advisability of using these media for the cultivation of P. aeruginosa microorganisms.

Differences between the number of microbial cells when using a combination of enhancers A, B, C in concentrations from 0.001 ± 0.0005% to 0.1 ± 0.05% and control are statistically significant. This indicates the effectiveness of the use of growth stimulants of various concentrations in combination to increase the number of microbial cells.

In the table. Figure 7 shows the exposure of the appearance of P. aeruginosa colonies on different media without growth stimulators.

Table 7 .- Exposition of the appearance of colonies of P. aeruginosa on various media at a sowing dose of 10 ⁶ -

Exposure, hours Number of colonies, CFU / ml V, colonies / hour.

12 2 * 0.16

6 ** 0.5

20 *** 0.83

18 0.4 x 10 ² * 2.2

Note: * - nutrient agar, ** - erythrocyte mass, *** - grain bard

As can be seen from the data table. 7, colonies of microorganisms appeared after 12 hours of incubation: on the nutrient agar - 2 colonies, on the erythrocyte mass there were 6 colonies, and on the grain bard - 10 colonies. After 18 hours of incubation, 40 colonies were observed on nutrient agar, 60 colonies on erythrocyte masses, and 100 colonies on grain bard.

That is, the largest number of colonies was observed after 18 hours of incubation and amounted to 10 ² CFU / ml, and the highest reproduction rate was 5.5 col. / hour

The use of enhancers influenced the appearance of colonies (Table 7). At a concentration of 0.1 ± 0.05% A, Pseudomonas aeruginosa colonies appeared after 10 hours of incubation, whereas on control media after 12-14 hours. A concentration of 0.01 ± 0.005% A decreased the cultivation period and colonies of microorganisms were recorded after 6-8 hours. The addition of 0.001% concentration of stimulant A to the medium facilitated the appearance of colonies after 7 hours. There were no particular differences in incubation time between enhancer concentrations of 0.01 ± 0.005% and 0.001 ± 0.0005% for the appearance of colonies. The difference in the number of colonies between growth stimulants A, B, and C at different incubation periods did not significantly differ.

Table 8 .- Exposition of the appearance of P. aeruginosa colonies on culture media using enhancers and the rate of reproduction at a seeding dose of 10 ⁶

12 102 * 8.3

3 x102 ** 8.3

102 *** 8.3

18 103 * 55.5

5x103 ** 278

103 *** 55.5

Note: * - nutrient agar, ** - erythrocyte mass, *** - grain barda According to the data in the table. 8, when enhancers were added at a concentration of 0.1 ± 0.05% A, colonies appeared after 10 hours of incubation, whereas on control media after 12-14 hours. The number of colonies after 10 hours of incubation was: 20 colonies on nutrient agar, 31 colonies on red blood cells and 40 colonies on grain bard. After 11 hours of incubation: 100 colonies on nutrient agar, 45 colonies on erythrocyte mass, 100 colonies on grain bard. After 12 hours of incubation, more than 100 colonies were observed on all of these media. After 18 hours of incubation, the number of colonies was - 10 ³ on all of the above media.

That is, the largest number of colonies with the addition of enhancer A at a concentration of 0.1% was observed after 18 hours of incubation and amounted to 5 x 10 ³ CFU / ml, and the highest reproduction rate was 278 colonies / hour.

In the table. Figure 9 shows how growth stimulants at a concentration of 0.01% A affect the appearance of colonies on various nutrient media (nutrient agar, erythrocyte mass, and grain bard). Table 9. - Exposition of the appearance of colonies on nutrient media with the addition of enhancers at a concentration of 0 , 01 ± 0.005% A at a sowing dose of 10 '

Exposition, hour enhancers Number of colonies, V, colonies / year.

CFU / ml

6 0.01 ± 0.005% 4 * 0, 7

A 6 ** 1.0

7 *** 1.17

7 * 0.57

6 ** 0.86

g% ** 1.14 8 8 * 1,0

10 ** 1.25

g *** 1.0

9 14 * 1.5

16 ** 1.8

18 *** 2.0

10 24 * 2.4

36 ** 3.6

38 *** 3.8

1 1 98 * 8.9

57 ** 5.2

60 *** 5.4

12,148 * 12.3

7.5

100 *** 8.3

18 106 * 5.5 x 104

4 x 106 ** 22 x 104

5 x 106 *** 27 x 104

Note: * - nutrient agar, ** - erythrocyte mass, *** - grain bard

As the data in table. 9, with the addition of enhancers at a concentration of 0.1 ± 0.05%, regardless of the type of enhancer, colonies were recorded after 6-8 hours of incubation, whereas on control media after 12-14 hours. The number of colonies after 6-7 hours of incubation was: 4 colonies on nutrient agar, 6 colonies on erythrocyte mass, 7 colonies on grain bard. After 10 hours of incubation: 24 colonies on nutrient agar, 36 colonies on red blood cells and 38 on grain bard. After 11 hours of incubation: 98 colonies on nutrient agar, 57 colonies on erythromass and 60 colonies on grain bard. After 12 hours of incubation, 148 colonies were recorded on nutrient agar, 90 colonies on erythromass, and 100 colonies on grain bard. After 18 hours of incubation, 106 colonies were observed on all of the above media. That is, the largest number of colonies with the addition of enhancers A at a concentration of 0.01 ± 0.005% was observed after 18 hours of incubation and amounted to

B x 10 ⁶ CFU / ml, and the highest reproduction rate was 27 x 10 colon / hour.

As shown in the table. 10, there were no special differences in incubation time between enhancer concentrations of 0.01 ± 0.005% and 0.001 ± 0.0005%. At the time, the appearance of colonies was not observed. When enhancers were added at a concentration of 0.1 ± 0.05% A, colonies appeared after 7 hours of incubation, whereas on control media after 12-14 hours. The number of colonies after 7-8 hours of incubation was: 9 colonies on nutrient agar, 10 colonies on red blood cells and 8 colonies on grain bard. After 11 hours of incubation: 100 colonies on nutrient agar, 60 colonies on erythromass and grain bard. After 12 hours of incubation, 150 colonies on nutrient agar, 100 colonies on red blood cells and 160 colonies on grain bard. After 18 hours of incubation, 106 colonies on all media.

That is, the largest number of colonies with the addition of enhancer A at a concentration of 0.001% was observed after 18 hours of incubation and amounted to 6 x 10 ⁶ CFU / ml, and the highest reproduction rate was 33.3 x 104 colonies / hour.

Table 10.- Exposition of the appearance of colonies on nutrient media with the addition of enhancers at a concentration of 0.001% A at a sowing dose of 10 ⁶

Exposition, hour enhancers Number of colonies, V, colonies / year.

CFU / ml

8 0.001 ± 0.0005 * 1.12

% A 10 ** 1.25

- - g *** 1,0

9 15 * 1.6

16 ** 1.7

17 *** 1.8

10 20 * 2.0

30 ** 3.0

41 *** 4.1 11 102 * 9.09

60 ** 5.4

90 ** 8.2

12 150 * 12.5

102 ** 8.3

160 *** 13.3

18 106 * 5.5 x 104

106 * 5.5 x 104

6 x 106 *** 33.3 x 104

Note: * - nutrient agar, ** - erythrocyte mass, *** - grain bard

Example 2. - Reduction of virulence of P. aeruginosa under the influence of growth enhancers on the example of the suppression of adhesive properties

It is known that the adhesion of microorganisms is the first stage of colonization, the main and determining factor of their virulence and pathogenicity. Using adhesins, microbes recognize receptors on cell membranes, attach to them and colonize various surface structures of the cell wall. The ability of bacteria to adhesion and colonization of surfaces is fixed by natural selection. This function is necessary for bacteria with saprophytic existence. For example, legionella actively attach to the surface of cyanobacteria, cholera vibrios actively colonize zooplankton, the chitin of which they use as a food source and stimulates the multiplication of cholera vibrios [ ⁴ ]. The study of the adhesion of microorganisms is of particular importance for medical microbiology, which has received clinical confirmation: it has been established that in the absence of adhesins, neither bacteria nor fungi can grow and form colonies, and if there is no 1 shunization then there is no infection and disease [ ⁵ ].

Adhesion of a bacterial pathogen can be carried out to the components of the extracellular matrix — fibronectin, collagen, laminin, etc. Matrix proteins have an RGD sequence with which integrins of the cell surface interact. Thus, extracellular matrix proteins contribute to the adhesion of bacteria to the target cells of the host. The adhesion of bacteria to such proteins is specific the nature and each pathogen implements this opportunity in its own way. For the manifestation of the pathogenicity of some bacteria, their interaction with matrix proteins is critical.

Most gram-negative bacteria attach to epithelial cells of humans and animals using adhesins, which are special organelles [ ⁶ ]. Many pathogenic microorganisms are able to penetrate the host cells and proliferate actively in them. Adherent molecules called invasinams are used to penetrate bacteria into cells. The most common mechanism involves the activation of signals in the host cell, allowing the invasion of bacteria by triggering normal cellular responses.

Considering the presence of substances that can affect the manifestation of adhesion, it is possible to direct their action to prevent the development of an infectious process []. One way to block the adhesion mechanisms is to use antibacterial drugs in low concentrations that inhibit the process of fixing pathogens in the primary infection zone [ ⁸ ]. For this purpose, specific bacteriophages can also be used [ ⁹ ]. To determine the adhesive properties of microorganisms, the most convenient model in which human red blood cells are used as macroorganism cells. An increase in cell biomass under the influence of enhancers leads to a change in some biochemical tests. One of them is the adhesive properties of microorganisms (table. 1 1)

Table 1 1 .- Comparative Adhesive Properties (IA) in P.aeruginosa

Enhancers Adhesion Index (IA)

Pseudomonas Pseudomonas Pseudomonas

aeruginosa aeruginosa aeruginosa

ATCC 27853 ATCC 9027 12-76

0.01 ± 0.005% A 2.6 ± 0.3 * 3.4 ± 0.2 * 3.5 ± 0.3 *

0.01 ± 0.005% V 2.5 ± 0.2 * 3.5 ± 0.2 * 3.5 ± 0.3 *

0.01 ± 0.005% C 2.6 ± 0.2 * 3.3 ± 0.2 * 3.6 ± 0.3 *

0.001 ± 0.0005% 2.6 ± 0.3 * 3.4 ± 0.1 * 3.7 ± 0.4 *

BUT

0.001 ± 0.0005% 2.7 ± 0.4 * 3.6 ± 0.3 * 3.8 ± 0.3 *

AT 0.001 ± 0.0005% 2.4 ± 0.4 * 3.5 ± 0.3 * 3.7 ± 0.3 *

FROM

0.01 ± 0.005% A 1.4 ± 0.3 * 1.5 ± 0.3 * 1.4 ± 0.3 *

0.01 ± 0.005% V

0.01 ± 0.005% C

0.001 ± 0.0005 1.8 ± 0.2 * 1.9 ± 0.4 * 1.7 ± 0.4 *

% A

0.001 ± 0.0005

% B

0.001 ± 0.0005

% C

KOH- 3.2 ± 0.3 3.1 ± 0.3 3.2 ± 0.3

troll

Note: * - the difference in indicators is statistically significant (p <0.05)

As evidenced by the data on the determination of the degree of adhesion by IA during the cultivation of the microorganism P. aeruginosa on media with enhancers, which are given in table. 11, adhesion indices differed from control indices. Strains that were moderately adhesive retained these properties upon cultivation with growth stimulators separately. Separate use of enhancers at a concentration of 0.001 ± 0.0005% reduced the adhesive activity of Pseudomonas aeruginosa strains to (2.4 ± 0.4) - (2.7 ± 0.4), and the strains became low-adhesive. The combination of enhancers at a concentration of 0.001 ± 0.0005% contributed to a decrease in the adhesive activity of Pseudomonas aeruginosa strains to (1, 4 ± 0.3) - (1.8 ± 0.4). Medium-adhesive strains under the influence of growth stimulants became low-adhesive. Adhesion index was (1.4 - 1, 7) against (3.1 - 3.2) when grown on medium ^ ^{^} es adding nemetabolytng growth stimulants.

As a result of statistical data processing table. 11, it was shown that the differences between the adhesion indices of the strains cultured on media with growth stimulants A, B, C separately at a concentration of from 0.001 ± 0.0005% to 0.01 ± 0.005% or in combinations, and statistically controlled significant. This indicates an effective the use of enhancers A, B, C in a concentration of from 0.001 ± 0.0005% to 0.1 ± 0.05% to reduce the adhesive activity of microorganisms.

Example 3

Patient N., 42 years old, suffering from an open form of pulmonary tuberculosis caused by multidrug-resistant mycobacterium tuberculosis. The first and second line anti-TB drugs were ineffective (they used traditional therapy according to WHO criteria: isoniazid (H), rifampicin (R), pyrazinamide (Z), streptomycin (F)). The patient was repeatedly operated on. The patient was prescribed intravenous drip papaverine 1% 2 ml, dibazole 2% 2 ml and dipyridamole 0.5% 4 ml in one dropper per 400 ml of 0.9% sodium chloride solution intravenously dropwise once a day. On the third day of applying the mixture, a decrease in cough and pain was observed, and after 15 days on the Rq-gram, only residual tuberculosis phenomena in the form of calcifications were observed, according to clinical and microbiological data, remission began. During the year, upon examination of the patient, a relapse of the disease was not observed. Objectively: Rq-remission, mycobacteria-, Μ-, R-.

Example 4

Patient C, 70 years old, was hospitalized with a diagnosis of open tuberculosis of the upper lobe of the right lung, destruction +, mycobacteria +, M +, +, resistance +. Previously, the patient received treatment with isoniazid (H), rifampicin (R), pyrazinamide (Z), streptomycin (F), which was ineffective. The patient was given the same regimen (H, R, Z, F), but papaverine 1% 2 ml, dibazole 2% 2 ml and dipyridamole 0.5% 4 ml in one dropper per 400 ml were added 1 time per day. 0.9% sodium chloride solution is administered intravenously slowly once a day. The first signs of clinical improvement were observed after 4 days in the form of a decrease in the intensity of cough and pain, as well as a decrease in sputum separation. After 14 days, an Rq examination established remission, which lasts 12 months. Objectively:, Rq-remission, mycobacteria-, Μ-, R-.

Industrial applicability

The invention relates to microbiology, namely biotechnology, pharmacy and medicine and can be used to accelerate the growth of biomass probiotics, genetically engineered drugs, yeast, vaccine strains of microorganisms, lactic acid starter cultures, and can also be used to suppress the virulence of bacteria and fungi in the treatment of infectious diseases of humans and animals.

Information Sources Used

1. Glick B. Molecular butechnopa [Text] / B. Glick, J. Pasternak. - M .: Mir, 2002 .-- 1 15 p.

2. The use of active frequencies of electromagnetic radiation of millimeter and centimeter ranges in microbiology / A.Kh. Tambiev, N.N. Kirikova, A. A. Lukyanov // High technology. - 2002- N ° l. - S.26-33

3. Vityuk N.V. Analysis of the structure-activity relationship of clonidine-like imidazolines based on the above description of the molecular structure [Text] / N.V. Vityuk // Chem. journal - 1997.- t.31. - JY ° 4. - S.44-47

4. US Patent 4997765 (Microorganism growth acceleration) Gary W. Evans, Filed Jul. 29, 1988, Date of Patent Mar. 5, 1991

5. US Patent Application Publication US2010 / 0249234 Al Sep. 30, 2010 (Methods of reducing virulence in bacteria) Ching-Hong Yang

6. Adhesive and some other properties of Vibrio cholerae ТСР + СТХ- isolated from environmental objects of the Rostov region in 2002 / N. R. Telesmanich, Yu.

L. Lomov, I. Kh. Bardykh [et al.] // Journal of Microbiology, Epidemiology and Immunology

7. Kawasaki Y. Inhibitory effects of bovine lactoferrin on the Adherence of enterotoxigenic Escherichia coli to host cells / Kawasaki Y., Tazume S., Shimizu K., Matsuzawa H., Dosako S., Isoda H., Tsukiji M., Fujimura R., Muranaka Y., Isihida H. // Biosci-Biotechnol-Biochem. - 2000. - Vol. - 64. - JN ° 2. - P. 348 - 354.

8. Nikolaev A. Yu. Adhesion and release agent Pseudomonas fluorescens / A. Yu. Nikolaev, D. I. Prosser // Journal of Microbiology, Epidemiology and Immunology. - 2000. - T. 69, J ° 2. - S. 237 - 242.

9. Welty W. K. Proinflammatory cytokines increase in sepsis after anti-adhesion molecule therapy / Welty WKE, Carraway MS, Ghio A., Kantrow SP, Huang Y. C, Piantadosi CA // Shock. - 2000. - Vol. 13. -tfs 5.- P. 404-409. 10. Vranes JJ Effect of subminimal ingibitory concentration of azithromycin on adherence of Pseudomonas aeruginosa to polystyrerene / Vranes JJ // Chemother. - 2000. - Vol. 12. -

1 1. Aslanov B. I. Justification for the use of a bacteriophage to combat

Pseudomonas aeruginosa infection in a trauma hospital: abstract. dis. to receive scientific Candidate of medical sciences: special. 07/03.00 "GuPkrobyulopya" / B. I. Aslanov. - St. Petersburg, 2001 .-- 24 p.

Claims

Claim

I. A method for accelerating the growth of bacterial biomass and suppressing virulence of bacteria, characterized in that phosphorylation inducers of intracellular proteins are used as inhibitors of virulence of microorganisms.

2. The method according to claim 1, characterized in that cyclic adenosine monophosphate accumulation activators are used as inducers of phosphorylation of intracellular proteins.

3. The method according to claim 2., Characterized in that as a mixture of activators of the accumulation of cyclic adenosine monophosphate use papaverine, bendazole and dipyridamole.

4. The method according to claim 3, wherein papaverine, bendazole and dipyridamole are in the form of salts.

5. The method according to claim 3, wherein papaverine, bendazole and dipyridamole are in the form of bases.

6. The method according to p.Z., characterized in that the ratio of papaverine, bendazole and dipyridamole are in the range of concentrations in solution from 0.0001 ± 0.00005 g /% to

2.0 ± 0.05 g /% each

7. The method according to claim 6, characterized in that in order to suppress the virulence of the bacteria, the infectious patient is prescribed a mixture of papaverine, bendazole and dipyridamole according to claim 6. 1-10 days before the appointment of an antibiotic or in parallel with it.

8. The method according to claim 6., Characterized in that papaverine, bendazole and dipyridamole are administered parenterally to the patient.

9. The method according to claim 6., Characterized in that papaverine, bendazole and dipyridamole are administered to the patient orally.

10. The method according to claim 6, characterized in that papaverine, bendazole and dipyridamole are administered rectally to the patient.

I I. The method according to claim 6, characterized in that papaverine, bendazole and dipyridamole are applied to the patient in the affected area with burns and infected purulent wounds.

12. The method according to claim 6., Characterized in that any antibiotic to which microorganisms are sensitive is used as an antibiotic.

13. A method of accelerating the growth of bacterial biomass and suppressing virulence of bacteria by adding low molecular weight growth accelerators to the medium, characterized in that a mixture of intracellular protein phosphorylation inducers is used as low molecular weight growth accelerators of microorganisms

14. The method according to item 13., Characterized in that cyclic adenosine monophosphate accumulation activators are used as inducers of phosphorylation of intracellular proteins.

15. The method according to 14., Characterized in that as a mixture of activators of the accumulation of cyclic adenosine monophosphate use papaverine, bendazole and dipyridamole.

16. The method of claim 15, wherein the papaverine, bendazole and dipyridamole are in the form of salts.

17. The method according to clause 15., Characterized in that the papaverine, bendazole and dipyridamole are in the form of bases.

18. The method according to p. 15., characterized in that the ratio of papaverine, bendazole and dipyridamole are in the range of concentrations in the nutrient medium from 0.0001 ± 0.00005 g /% to 2.0 ± 0.05 g /% each.