Method of determining nonspecific resistance of pathogenic microorganisms to antibiotics based on measuring catalytic activity of phosphodiesterases cleaving cyclic diguanosine monophosphate

FIELD: biotechnology.

SUBSTANCE: invention is a method of determining the nonspecific resistance of pathogenic microorganisms to antibiotics and the fact of the presence of bacterial biofilms on the basis of measurement of catalytic activity of phosphodiesterases cleaving the cyclic diguanosine monophosphate, with a threshold sensitivity of 50 pg/ml, comprising: 1) isolating the target-phosphodiesterase from lysed bacterial cells; 2) binding of phosphodiesterase with biotin-conjugated antibodies specific for non-catalytic domains of phosphodiesterase; 3) affinity purification of complexes formed by target-phosphodiesterase and biotin-conjugated antibody using paramagnetic particles containing neutravidin or its analogs that bind biotin; 4) interacting of the complexes of phosphodiesterase/biotin-conjugated antibody, immobilised on paramagnetic particles with complexes containing a-di-GMP in the form of G-quadruplex systems with intercalate dye, which is accompanied by decrease in the intensity while destruction of complexes of intercalate dye with c-di-GMP; 5) measurement of decrease of fluorescence upon hydrolysis with c-di-GMP and destruction of complex of c-di-GMP with intercalate dye, followed by quantitative estimation of the phosphodiesterase activity based on calibration curves made using known amounts of the recombinant enzyme of phosphodiesterase identical to the test target; 6) identification of increased level of phosphodiesterase activity detected by the test antibiotic-resistant bacterial strains capable of biofilm formation, as compared with the level of phosphodiesterase activity that can be detected for the control strains of bacteria of the same species not having the antibiotic resistance and the ability to form biofilms.

EFFECT: method enables to determine the nonspecific resistance of pathogenic microorganisms to antibiotics and to establish the fact of the presence of bacterial biofilms.

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The invention relates to biotechnology, specifically to the areas of Biomedicine, bioscreening and develop methods for diagnosis and monitoring of bacterial infections on the basis of the reporter biomolecules specific for the organism to the target, and relates to a new method of determining the presence of nonspecific antibiotic resistance caused by the formation of biofilms. These biofilms are changing the permeability of individual cells and cell colonies, hindering the penetration of antibiotics into the cell. In some cases, components of biofilms directly bind antibiotics, preventing their access to targets. Colonies of bacteria forming biofilms, have a high degree of adhesiveness as to a variety of surfaces, including medical instruments, such as the system of hemodialysis catheters and other equipment, and to the tissues of a living organism, such as various mucous membranes. Numerous studies have shown that one of the main problems in the treatment of chronic inflammatory infections is the high resistance of biofilms formed by pathogens-pathogens to antibiotics and other adverse environmental factors, as well as a high degree of adhesion of bacteria to the mucous membranes of the host body. The resistance of biofilms of Pseudomonas is one of the major reasons sky is Agapitova prognosis in patients with cystic fibrosis. The change in the balance of biofilm and planktonic state of the pathogen is the primary method of transmission of plague from fleas to mammals. There are a number of processes in bacteria, as partially studied, and still little studied, associated with the formation or dissociation of biofilms, as well as other switches bacterial phenotype, one way or another connected with the virulence of these pathogens and how to protect them from antibiotics and other adverse environmental factors. A Central role in the management of these processes plays a dinucleotide, cyclic degradationof (c-di-GMP), found only in bacterial cells. Synthesis and hydrolysis of c-di-GMP that occurs under the influence of external and internal signals, leads to changes in the concentration of this secondary vector in the cell. This causes global switching phenotype (e.g., planktonic forms to antibiotic-resistant biofilm and back), and modulation of various local processes, such as turning on and off the expression of different genes, reaction to light and chemicals, etc.

The main field of application technology detection of catalytic activity of phosphodiesterase c-di-GMP are clinical and biological research, Biomedicine and drug development.

Changed the e concentrations of c-di-GMP is controlled by two types of enzymes. Synthesis of c-di-GMP comes from GTP and is catalyzed by various degranulation, which have, as a rule, one or more regulatory domains, and are also regulated by the inhibition of the reaction product. The hydrolysis of c-di-GMP is under the influence of the family-specific phosphodiesterase, as well as degeneracies consisting of various regulatory domains and vysokogomogennogo catalytic domain (EAL domain, so named for the most conservative amino acid sequence). Currently known a number of processes that depend on changes in phosphodiesterase activity of c-di-GMP and directly associated with virulence. In particular, increased activity of phosphodiesterase leads to the increase of antibiotic resistance strains of Pseudomonas aeruginosa causing infection in cystic fibrosis (Hoffman LR, D'argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI (2005) Nature, 436, 1171-1175). The phosphodiesterase activity is directly related to the virulence of the plague pathogen (Bobrov AG, Kirillina O, Ryjenkov DA, Waters CM, Price PA, Fetherston JD, Mack D, Goldman WE, Gomelsky M, Perry RD (2011) Environ Mol, 79, 533-551). The causative agent of borreliosis production of c-di-GMP critical to maintain the life of the pathogen in the body transmitters. The causative agent of cholera uses c-di-GMP and the appropriate phosphodiesterase to switch between phenotype and activate the mechanisms of virulence. It is obvious that in the future the m list phosphodiesterase c-di-GMP will be updated. Determination of the activity of a particular phosphodiesterase will be crucial to determine the presence of a pathogen, his analysis of the phenotype and response to the effects of antibiotics. This can be very effectively used in the treatment of both chronic and especially dangerous infections, with the rapid determination of genotypes and phenotypes pathogens, components of the bacterial film on selection of inhibitors of phosphodiesterase c-di-GMP.

The known method of morphological analysis of the phenotype of bacteria forming biofilms, in particular biofilms formed by pathogenic Pseudomonas isolated from patients with cystic fibrosis (Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. (2006) Indian J Environ Med, 24, 25-29). There are also microscopic methods for the determination of the formation of biofilms as Pseudomonas and other pathogens (Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Braz J (2011) Infect Dis, 15, 305-311). These methods are used to assess the phenotype of the organism and its potential virulence and resistance to antibiotics, disinfectant agents and other factors. However, these methods cannot solve the problem of quantifying the production of proteins that cause certain virulence determinants, to determine the dynamics of changes in the level of non-specific resistance to antibiotics or to assess the effect of therapeutic inter is entii. Microscopic and morphological methods require a long time (several days), which is necessary for growth of colonies of microorganisms and development of diagnostic phenotype, usually resulting from the synthesis of extracellular polysaccharide matrix. In addition, phenotypic changes can occur in several scenarios and do not necessarily expressed in the change of antibiotic resistance.

Known methods for differential staining of bacteria forming biofilms, using dyes crystal violet on the bacteria attached to the high adhesive plastic (O'toole GA (2011) J Vis Exp, 47, pii: 2437), and Congo red, made in the agar medium (Harrison-Balestra C, Cazzaniga AL, Davis SC, Mertz PM (2003) Dermatol Surg, 29, 631-635).

Known direct methods for the determination of resistance of microorganisms to antibiotics, in particular the method of inoculation on selective medium with different concentrations of the antibiotic (Kronvall G, Kahlmeter G, Myhre E, Galas MF (2003) Clin Environ Infect, 9, 120-132), capillary method of disks, saturated defined concentration of the antibiotic and molecular-genetic methods for detecting the presence of genes for resistance to a particular antibiotic (Woodford N, Sundsfjord AJ (2005) Antimicrob Chemother, 56, 259-261). These methods are useful for determining the specific resistance to antibiotics, due to the production specification the specific protein, inactivating the antibiotic or ensure its excretion from the cell. However, such methods are unable to assess the development of nonspecific resistance to the antibiotic caused by external lock permeability of cells in connection with the formation of biofilms. Cells, the potential of having such resistance under artificial cultivation will be in the planktonic phase and the transfer phase of the biofilm will die from significantly lower concentrations of antibiotics, reducing the reliability of the analysis. Quantitative detection of phosphodiesterase activity of c-di-GMP, which is responsible for switching the phenotype is much more adequate parameter describing the degree of development of nonspecific resistance to the antibiotic than the above methods. It allows you to directly monitor the activity of a key component of the regulation of the formation of antibiotic-resistant biofilms in response to increasing concentrations of the antibiotic in the environment.

Known method of detection of c-di-GMP in vivo, based on the production of intracellular fluorescent sensory protein (Rybtke MT, Borlee BR, Murakami K, Irie Y, Hentzer M, Nielsen THOSE, Givskov M, Parsek MR, Tolker-Nielsen T (2012) Appl Environ Environ, 78, 5060-5069). This method can be used to monitor the total concentration of c-di-GMP and balance the concentration of phosphodiesterase - degeneracies, however, it is inefficient the La to determine the concentration of catalytic activity specific phosphodiesterase, responsible for development of resistance to the antibiotic, or for a certain switching phenotype, acting as a virulence factor.

Also known is the closest method of detection, which consists in determining the number of c-di-GMP in vitro using fluorescent protein sensor (But CL, Koh SL, Chuah m l, Luo Z, Tan WJ, Low DK, Liang ZX (2011) Chembiochem, 12, 2753-2758). However, this method cannot be directly used to determine the switching phenotype and the development of nonspecific antibiotic resistance, because it does not focus on the catalytic activity of phosphodiesterase target responsible for a particular process in the pathogen. Additionally, if you consider using this method as only detecting module for determining the activity of phosphodiesterase, its disadvantage is the use of recombinant protein construct, which complicates and increases the cost of detection, especially when low concentrations detected phosphodiesterase requires the use of an excess of substrate.

The invention solves the task of developing a highly sensitive method for determining the presence of catalytically active phosphodiesterase c-di-GMP in advance of a particular type, responsible for the process of esterase c-di-GMP in cells infectious bacterial agent, of the Vedas is walking to the development of nonspecific resistance to the antibiotic, switching virulent phenotype and other changes that may affect the progress of the disease or to serve as indicators of the effectiveness of therapy, and is applicable for detection of phosphodiesterase c-di-GMP obtained from clinical specimens, the result of the cultivation of the pathogen in vitro, or isolated from the pathogen, found in the environment.

The problem is solved by creating a method of detecting activity of c-di-GMP fosfodiesterasa target using the formed complex G-quadruplexes c-di-GMP and fluorescent dye capable of intercalated into the structure of the G-quadruplexes c-di-GMP, increasing by intercalation, the intensity of the intrinsic fluorescence and reducing the intensity of intrinsic fluorescence in the destruction of G-quadruplexes due to the hydrolysis of c-di-GMP phosphodiesterase-target.

Also the problem is solved through the use of affinity probes in the form of monoclonal or polyclonal antibodies, or nucleic acid or peptide aptamers that are specific to one of the non-catalytic modules c-di-GMP phosphodiesterase in such a way that the binding of this module has no effect or negligible influence on the ability of the target to hydrolyze c-di-GMP.

Also the problem is solved through the use of a mixture of detergents and nucleases, providing soft lysis of bacterial cells and the destruction of high molecular weight the nucleic acid, secure the release into solution phosphodiesterase-targets for access to affinity probes.

Also the problem is solved at the expense of heterophase principle phosphodiesterase target, in which complex targets with affinity probes, specifically adsorbed on the solid phase, while the remaining components of the mixture are removed from the solution.

Also the problem is solved by creating the control of enzymes, representing a recombinant analogue of the analyzed targets with pre-calculated specific enzymatic activity.

And the problem is solved due to the effective quantification of the enzymatic activity of phosphodiesterase target immobilized on a solid phase using affinity probes, based on measurements of the fall of the fluorescence of the G-quadruplexes in comparison with the fall of the fluorescence in the control reactions performed with recombinant enzyme target.

The technical result of the invention is to provide a rapid and highly sensitive method for determining the concentrations and dynamics of concentration changes under the effect of therapy and other incentives or antagonists, key of c-di-GMP dependent regulators with the activity of c-di-GMP phosphodiesterase. The sensitivity of the developed technique depends on the catalytic effect is aktivnosti analyzed phosphodiesterase-target and average of 50 PG/ml of enzyme.

The proposed technical solution, as a substrate for determining the activity of c-di-GMP phosphodiesterase apply complex fluorescent intercalating dye, which is capable under certain conditions (high concentration of salts of alkali metals, the concentration of c-di-GMP, sufficient for the formation of G-quadruplexes (Nakayama S, Kelsey I, Wang J, Sintim HO (2011) Chem Commun, 47, 4766-4768). G-QUADRUPLEX - specific patterns, which form mainly of RIBO - and desoxyribonuclease goinnovate nucleotides. G-QUADRUPLEX are some of the most common structures, which is the intercalation of the fluorescent dyes (such as, in particular, SYBR Green), bind to the DNA and quantitative analysis of products developed PCR in real-time. Upon intercalation of the dye in DNA or RNA is a flare-up of fluorescence, while reducing the number of structures that can form G-QUADRUPLEX to decrease the intensity of fluorescence. The destruction of G-quadruplexes formed by c-di-GMP may occur, for example, due to the decrease of the concentration of this dinucleotide rod action-specific phosphodiesterase. It is important that other nucleases or phosphodiesterase unable to degrade c-di-GMP, which provides a high JV is civicnet of this method relative to the RBCs c-di-GMP. The preferred dyes are intercalating the azole type dyes, SYBR Green and its analogues. The most preferred intercalating in G-QUADRUPLEX dye for detection and quantitative determination of activity of phosphodiesterase is dye titlovi orange (Nakayama S, Kelsey I, Wang J, Roelofs K, Stefane B, Luo Y, Lee VT, Sintim HO (2011) J Am Chem Soc, 133, 4856-4864), which has one of the biggest values of changes in fluorescence intensity when changing the concentration of G-quadruplexes c-di-GMP. The principal advantage of using titlepage orange and other intercalating dyes in comparison with fluorescent analogs of c-di-GMP and fluorescently protein sensors c-di-GMP is a low cost and high stability of intercalation c-di-GMP when compared with other listed types of probes the values of the signal changes when analyzing the activity of phosphodiesterase.

The important condition of successful technical solution of the task of determining the activity of phosphodiesterase-targets in biological samples is the use of antibodies specific to the domain of phosphodiesterase c-di-GMP, other than catalytic, binding, which does not cause significant changes in catalytic efficiency of the enzyme target. The use of such antibodies allows you to specifically select a phosphodiesterase-mission is from the cell lysate without loss of catalytic activity, and analyze activity affine selected target.

A significant advantage of this method is the use of heterogeneous phase detection enable automated analysis method phosphodiesterase activity of c-di-GMP. At the same time, the use of heterophase selection provides a simple and effective allocation of individual RBCs target in the analysis process. The preferred matrices for holding heterophase analysis are paramagnetic microparticles coated with affinity ligands, providing specific allocation of complexes of antibody-phosphodiesterase from solution. Preferred ligands are proteins that bind immunoglobulin, such as protein A, protein G, and their analogues. The most preferred system for affinity adsorption of phosphodiesterase-targets are specific to the target antibodies modified with Biotin, paired with paramagnetism microparticles coated with a Biotin-binding protein. The most preferred protein for coating paramagnetic microparticles is neutravidin for the least non-specific adsorption of proteins from solution.

Automation of the method provides for its applicability for high-throughput screening of libraries of small organic molecules with the aim of obtaining inhibitors asset is barb fosfodiesterasa c-di-GMP, blocking the formation of biofilms, switching the phenotype of pathogens and the expression of virulence determinants. Automation also improves the reproducibility of the results of clinical and diagnostic research phosphodiesterase activity of c-di-GMP.

The advantages of the inventive method for the determination of nonspecific resistance to antibiotics and detecting the formation of biofilms include:

1) high sensitivity and specificity, based on the determination of the activity of the underlying determinants of these processes - regulatory phosphodiesterase c-di-GMP, but not indirect signs, such as the phenotype of the colonies, or staining with certain dyes; 2) the compatibility of the method with the technologies of real-time PCR and enzyme-linked immunosorbent assay, which is widely used in clinical diagnosis; 3) resistance to false-positive results, ensure specificity of hydrolysis of c-di-GMP affinity and specificity of adsorption specified phosphodiesterase-target drugs mono - or polyclonal antibodies; 4) the relative cheapness of the components of a diagnostic kit compared to recombinant fluorescent proteins and fluorescent and fluorogenic derivatives of c-di-GMP, the synthesis of which roads and remains difficult chemical problem.

The invention is carried out as follows

Carry out the preparation of G-quadruplexes c-di-GMP with intercalated thiazolium orange in solution with concentrace salts of alkali metals 0.3 M

Target fosfodiesterazu extracted from lysed bacterial cells by the method of affinity adsorption on paramagnetic particles coated neutravidin, using as intermediate affinity ligand biotinylated antibodies or other affinity molecule (e.g., aptamers)that are specific to the non-catalytic domains phosphodiesterase. The obtained immobilized immune complexes phosphodiesterase-target antibody is washed from unbound and low-affinity impurities and performed affinity purification of complexes formed by the target and biotinylating antibodies by the method of magnetic separation by binding biotinylated antibodies with paramagnetic particles coated neutravidin or its analogues that bind Biotin.

Microparticles with immobilized phosphodiesterase placed in a solution containing an excess of c-di-GMP in the form of G-quadruplexes carrying a fluorescent intercalating dye. Evaluate the activity of the enzyme based on the interaction of the complexes phosphodiesterase/biotinylated antibody immobilized on paramagnetic particles, complexes containing c-di-GMP in the form of G-quadruplexes with intercalated dye, measuring the fall of the fluorescence by the hydrolysis of c-di-GMP and the destruction of the fluorescent complexes of c-di-GMP with intercalating dye under de the action of phosphodiesterase. The concentration of fluorescent complexes calculated in advance based on the kinetic parameters of the hydrolysis of c-di-GMP for enzyme target, obtained in recombinant form using overproduction in E. coli. Qualitative and quantitative assessment of enzyme activity target is produced by comparing the curves obtained values fall fluorescence of complexes of c-di-GMP/intercalating dye with the calibration curves of the values fall fluorescence obtained for the known quantities are identical to the investigated target recombinant phosphodiesterase with known specific activity. Level phosphodiesterase activity detected by the tested bacterial strains used as criterion of high nonspecific antibiotic resistance and efficiency of the formation of biofilms in comparison with levels phosphodiesterase activity identified for the control of bacterial strains of the same species that does not have antibioticassociated and ability to form biofilms.

Schematic diagram of the embodiment of the invention shown in Figure 1. The invention is illustrated by the following graphics:

Figure 1. Scheme of the experiment for determination of the activity of phosphodiesterase c-di-GMP in solution on the basis of the detection of the fall of the fluorescent signal caused by the destruction of the complexes G-quadruplexes c-di-GMP with intercalating fluorescent dye.

Figure 2. The formation of complexes of G-quadruplexes c-di-GMP with intercalating dye thiazolium orange.

The figure shows the increase in fluorescence intensity upon addition of various concentrations of c-di-GMP to titlova orange (30 μm) in buffer containing 1 M sodium chloride and 20 mm Tris-HCl pH 7.5. The measurements were carried out on the device Varioskan Flash (Thermo, USA) at the ratio of the wavelengths of the excitation/detection 507/535 nm. X-axis presents the concentration of c-di-GMP: μm 1-0, 2-1 μm, 3-2 μm, 4-5 μm, 5-10 μm, 6-15 μm, 7-20 μm, 8-30 microns.

Figure 3. Detection of activity of phosphodiesterase arrR. Detection of activity of phosphodiesterase arrR was carried out in a solution containing 20 mm Tris-HCl, 300 mm potassium chloride, 3 mm magnesium chloride, 30 μm complexes titlepage orange with c-di-GMP. X-axis shows the different concentrations of phosphodiesterase arrR (ng/ml): 1-10, 2-4, 3-2, 4-1,5-0,4, 6-0,2, 7-0,1, 8-0,04, 9-0,02, 10-0.

Figure 4. A comparison of the level phosphodiesterase activity detected in strains of P.aeruginosa, the level of antibiotic resistance of the strains and the presence in the culture of bacterial biofilms. All bacterial cultures were grown in liquid medium under standard conditions for 12 hours.

On the X-axis in the figures presents: 1 - the bacteria P. aeruginosa strain ADS 27853 grown without added antibiotics; 2 - bacterium P. aeruginosa strain ADS 27853 grown in the presence of 8 μg/the l gentamycin; 3 - bacterium P. aeruginosa obtained from laboratory animals infected with different clinical isolates of the pathogen.

The invention is illustrated by the examples.

Example 1. Preparation of test samples phosphodiesterase arrR pathogen P. aeruginosa.

Cells of the pathogen obtained from model animals chronically infected with P. aeruginosa (Anderson GG, Moreau-Marquis S, Stanton BA, O'toole GA (2008) Infect Immun, 76, 1423-1433) and cells of P. aeruginosa (strain ADS 27853) are grown for 12 hours at 37°C in liquid culture on trypticase-soy broth without added antibiotics and in the presence of 8 μg/ml of the antibiotic gentamicin and determine the concentration of viable bacteria by plating on solid medium.

Grown bacteria washed twice by centrifugation in phosphate-buffered saline pH 7.3. The washed cells are lysed by the composition of B-PER (Thermo Pierce, USA)using the manufacturer's instructions with the addition of Dnesneho cocktail (Roche, USA) to reduce the viscosity of the solution. The lysate clarify by centrifugation and either used directly for analysis or frozen at -80°C, adding up to 1% trehalose and 10% glycerol as cryoprotectants.

Example 2. Formation and affinity purification of immune complexes phosphodiesterase arrR with biotinylating antibodies on paramagnetic particles with neutravidin to measure hydrolytic Akti the activity in relation to c-di-GMP.

To solubilization protein preparation obtained from cells of P. aeruginosa PAO1 add 100 ág/ml biotinylated polyclonal rabbit antibodies to extramembranous domain arrR (Gotoh H, Zhang Y, Dallo SF, Hong S, Kasaraneni N, Weitao. T (2008) Environ Res, 159, 294-302). After incubation for 2 hours to the lysate add 100 μl of the suspension of paramagnetic particles loaded with neutravidin (Thermo Pierce, USA), and incubated for 30 min with shaking. After incubation, the mixture is washed 10 times with phosphate-saline buffer pH 7.3 (2 ml)containing 20 μg/ml acetylated bovine serum albumin, containing by or nuclease activity, and 0.05% Triton X-100, required to maintain the transmembrane domain arrR in solubilization condition.

The washed microspheres containing immune complex arrR, mixed with a solution.

Example 3. Obtaining complexes of G-quadruplexes c-di-GMP with intercalating dye for measuring the activity of phosphodiesterase.

Cyclic dinucleotide at a concentration of 50 µm (2-fold excess of the minimum excess substrate, calculated for arrR) is mixed with a buffer containing 300 mm potassium chloride, 10 mm potassium phosphate pH 7.5. The mixture is heated to 95°C for 10 minutes and slowly cooled to room temperature (30 minutes, forced cooling programmable thermostat), then add titlovi orange to a concentration of 300 μm. The resulting mixture is incubated at a temperature of +4°C over night. The completeness of the formation of complexes determine fluorimetrically on the basis of standard calibration curves. Values of molar extinction for c-di-GMP and dye are equal 21600 and 63000 M-1cm-1respectively.

Example 4. Definition phosphodiesterase activity arrR splitting complexes G-quadruplexes c-di-GMP with intercalating dye.

To determine phosphodiesterase activity arrR paramagnetic microparticles coated neutravidin with immobilized complexes phosphodiesterase arrR/biotinylated antibody is titrated in the microplate for fluorescence measurements in dilutions 1:2, 1:5, 1:10, 1:50, 1:100, 1:500, 1:1000. Titrated microparticles are separated from the buffer by means of the magnet and to the microparticles with immobilized phosphodiesterase type complexes G-quadruplexes c-di-GMP with intercalating dye. The microplate is incubated in the chamber of the scanning fluorimetry 2 hours at 37°C, holding the measurement of fluorescence. The wavelength of excitation when the measurement is 508 nm, the fluorescence measurement is integrated in the range 518-700 nm. The obtained data are compared with values falling fluorescence measured at the same time with known quantities of recombinant phosphodiesterase arrR, taken in a series of concentration is, appropriate dilutions of a solution with a concentration of 20 ng/ml 1:2, 1:5, 1:10, 1:20, 1:50, 1:100, 1:200, 1:500, 1:1000. The sensitivity of the method definition phosphodiesterase activity on the basis of the control dimension is 50 PG/ml phosphodiesterase Activity in the positive control sample derived from the lysate of bacteria P. aeruginosa strain ADS 27853 grown without antibiotics, corresponds to the activity of the recombinant enzyme arrR, taken at a concentration of 200 PG/ml, and grown in the presence of gentamicin - 2 ng/ml phosphodiesterase Activity in samples obtained from model animals chronically infected with P. aeruginosa, corresponds to the activity of the enzyme arrR at a concentration of 100 PG - 4 ng/ml.

Example 5. Determination of antibiotic resistance of the tested bacteria and the presence of bacterial biofilms in bacterial cultures

Using grown 12-hour culture, determine antibioticassociated bacteria P. aeruginosa (strain ADS 27853 and bacteria derived from animal models) seeding trypticase-soy agar containing different concentrations of aminoglycoside antibiotic gentamicin (4, 8, 16, 32 and 64 μg/ml of the antibiotic). For culture of the strain of P. aeruginosa was ATSS 27853 grown in the absence of gentamicin, resistance to antibiotics is 4 µg/ml, and the culture of the same strain grown with add is the group of gentamicin, resistance to antibiotics is 32 µg/ml Bacterial cultures obtained from infected animals, resistance to gentamicin at a concentration of 4-64 mg/ml in accordance with detectionin the level of activity of phosphodiesterase arrR.

Biofilm formation in 12-hour cultures of P. aeruginosa detected microscopically and evaluate the results of spectrophotometric measurements of bacterial suspension (λ=575 nm), stained for 15 minutes with 1% crystal violet solution, followed by washing and solubilization of the dye in 96% ethanol, and the number of viable cells detected by titration method on cups. Culture of a strain of P. aeruginosa was ATSS 27853 grown for 12 hours in the absence of gentamicin, characterized by lower viability than the culture of the same strain grown with the addition of gentamicin, which shows the effective formation of biofilms by bacteria in the presence of the antibiotic. Bacterial cultures obtained from infected animals, find the efficiency of the formation of biofilms, rising in accordance with detectionin the level of activity of phosphodiesterase arrR.

The method of determination of non-specific resistance of pathogens to antibiotics and the presence of bacterial biofilms on on the basis of the measurement of catalytic activity of phosphodiesterase, breaks down cyclic degradationof, with a threshold sensitivity of 50 PG/ml, including:
1) allocation of phosphodiesterase target from lysed bacterial cells;
2) binding phosphodiesterase biotinylating antibodies specific to non-catalytic domains phosphodiesterase;
3) affinity purification of complexes formed by phosphodiesterase-target and biotinylated antibody using paramagnetic particles containing neutravidin or its analogs that bind Biotin;
4) interaction complexes phosphodiesterase/biotinylated antibody immobilized on paramagnetic particles, complexes containing C-di-GMP in the form of G-quadruplexes with intercalated dye, accompanied by a drop in fluorescence intensity as the destruction of the complexes, intercalating dye c-di-GMP;
5) measurement of the fall of the fluorescence by the hydrolysis of c-di-GMP and the destruction of the complex c-di-GMP with intercalating dye with subsequent quantitative determination of the activity of phosphodiesterase on the basis of the calibration curves constructed using known amounts of recombinant enzyme phosphodiesterase, identical to the investigated target;
6) identification of high level phosphodiesterase activity detected by testing the antibiotic-resistant bacteria the financial strains, is capable of forming biofilms, compared with the level phosphodiesterase activity detected for the control of bacterial strains of the same species that does not have antibioticassociated and ability to form biofilms.


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2 ex

FIELD: medicine.

SUBSTANCE: method of simultaneous immunochromatography analysis of PSA and CEA oncoantigens is offered. The analysis involves using a test strip which contains three segments A, B and C; the segment A overlaps 1-2 mm of the segment B. The segment A represents an inert porous carrier made of fibre glass ("ПЭД") with two reaction zones 1 and 2 applied on its surface. Mice monoclonal PSA and CEA antibodies conjugated with colloidal gold are respectively applied on zones 1 and 2. The conjugates are applied in the form of parallel strips in the centre of "ПЭД" perpendicularly to a fluid flow. The segment B represents nitrocellulose immobilised on a lavsan substrate with two test zones applied on its surface (monoclonal PSA and CEA antibodies in each), and a reference area (mice immunoglobulin antibodies).

EFFECT: test enables the simultaneous detection of the patients with the high blood serum PSA and CEA antigen contents in screening assays.

6 ex, 4 dwg

FIELD: medicine.

SUBSTANCE: analysed sample is studied simultaneously by two methods: the first one is a indirect immunofluorescence (IIMF) reaction, while the second one involves a polymerase chain reaction (PCR); the IIMF provides using monoclonal antibodies "BCKK" (P-384D and 434D). The antibodies interact with the capsular antigen F1 specific for Y.pestis species, or the plasmid-temperature-independent surface protein PFV which is found in all Y.pestis strains and rare R-form Y.pseudotuberculosis strains. The bacteria luminescence shows the presence of native or fraction-less Y.pestis bacteria, or typical and PFV-atypical Y.pseudotuberculosis strains in the sample. The PCR is conducted by two pairs of primers vlm33for/ISrevl754 - specific for Y.pestis species, and JS - specific for Y. pseudotuberculosis species. The values derived with the first pair of the primers are estimated as positive if observing the amplicons in 400 bps, and with the second pair if observing the amplicons in 223 bps; the analysed sample is identified by the matching the IIMF and PCR values with the reference strains.

EFFECT: method provides quick identification and high-reliability differentiation of all strains.

7 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: set of reagents for quantitative determination of avermectins contains an ivermectin conjugate with bovine serum albumin which is immobilised on a polystyrene dish, a peroxidase conjugate of highly specific mouse monoclonal antibodies to ivermectin, and ivermectin calibration samples (with concentration between 0 and 100 ng/ml). The monoclonal antibodies in the set are produced by a strain of hybrid cultured cells of Mus. Museums L., which are deposited in the Collection of transferred vertebrate somatic cells of the D. I. Ivanovsky Research Institute of Virology under No. 05/09. From non-specific components, the set contains a buffer for culturing the monoclonal antibody conjugate with horseradish peroxidase, a buffer for culturing the analysed samples and washing the trays, reagents for detecting peroxidase activity, a substrate solution, hydrogen peroxide and tetramethylbenzidine and a stop solution (1M sulphuric acid). The disclosed set is designed to detect residual quantities of avermectins in tissue and biological fluids for monitoring chemical contamination of animal products.

EFFECT: use of the set enables to determine main components of an avermectin complex in animal products, while providing high sensitivity and specificity of detection.

1 dwg, 2 tbl, 4 ex

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to immunology and biotechnology. The invention discloses a monomer single-strand V93 nanoantibody which can bind and inhibit the human vascular endothelial growth factor. The invention describes a nucleotide sequence which codes the V93 nanoantibody and its expression vector with extra epitope(s) on the C-end for detection and extraction and a signal peptide on the N-end. The invention discloses a method of obtaining the V93 nanoantibody, a method of inhibiting proliferation of endothelial cells using the V93 nanoantibody, as well as use of the V93 nanoantibody for qualitative and quantitative determination of VEGF in a sample.

EFFECT: use of the invention provides high-affinity neutralising monovalent single-strand nanoantibodies which are more resistant to external factors (temperature, pH) and cheaper to produce compared to conventional VEGF antibodies, which can be useful in medicine for treating and diagnosing diseases associated with regulation of the activity of the vascular endothelial growth factor (VEGF).

7 cl, 7 dwg, 6 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine, pharmacology, to methods and PTEN phosphatase inhibitor compositions for ovarian follicle and oocyte development in vitro. Using the PTEN phosphatase inhibitors, such as complexes of oxovanadate and peroxovanadate: bisperoxo (bipyridine) oxovanadate, bisperoxo (1,10-phenanthroline) oxovanadate, bisperoxo (picolinato) oxovanadate, bisperoxo (5-hydroxypyridine-2-carboxyl) oxovanadate, di- (picolinat) oxovanadate, di-(3-hydroxypicolinat) oxovanadate, bisperoxo (phenylbiguanide) oxovanadate, di-(phenylbiguanide) oxovanadate and bisperoxo (isoquinoline carboxylic acid) oxovanadate, provide the development and activation in vitro of oocytes and follicles, such as primordial, intermediate and primary ovarian follicles.

EFFECT: invention provides the development and activation in vitro of oocytes and follicles, such as primordial, intermediate and primary ovarian follicles.

14 cl, 3 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: composition contains: disubstituted potassium phosphate and hydrochloric acid - as buffer mixture components, water, a substrate or a combination of substrates selected from a group comprising pteridines, aldehydes, cysteine, and further contains an acrylic, olefin or siloxane polyether heteropolymer. The composition is used in clinical laboratory diagnosis and biochemistry to conduct a xanthine oxidase reaction.

EFFECT: easier conduction of the reaction, low toxicity of the composition and enabling serial manufacture thereof.

1 ex

FIELD: medicine.

SUBSTANCE: method for evaluating a degree of cardiomyocyte cytolysis accompanying infectious myocardial injuries consists in examining patient's blood plasma for activities of cardiospecific MB fraction of creatine phosphokinase, α-hydroxybutyrate dehydrogenase or the first and second fractions of lactate dehydrogenase, aspartate transaminase and alanine transaminase. A cardiomyocyte cytolysis index is calculated by formula: CCI=(MB-CPK/MB-CPKN·(α-HBDG/α-HBDGm)·(0.66 (AST/ALT)). The cardiomyocyte cytolysis index is used to evaluate the degree of cardiomyocyte cytolysis accompanying infectious myocardial injuries.

EFFECT: method enables describing the nature of enzymes, evaluating the degree of the change manifestations that is needed to specify the approach to the patient.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: activity of blood serum alkaline phosphatase (AP) and tartrate-resistant acid phosphatase (TRAP) is measured before the beginning and on 7-10th day of distraction in the patients undergoing surgical lengthening of extremity bones by Ilizarov's technique. The AP/TRAP relation is calculated. If the calculated AP/TRAP relation on the 7-10th day of distraction is within 120-150% with respect to the pre-operative value, no disturbed osteoreparative processes accompanied lengthening of extremity bones by Ilizarov's technique are stated in the patients. If the calculated AP/TRAP relation on the 7-10th day of distraction is less than 120% or more than 150% with respect to the pre-operative value, disturbed osteoreparative processes accompanied lengthening of extremity bones by Ilizarov's technique are stated in the patients.

EFFECT: improved assessment accuracy.

3 ex

FIELD: medicine.

SUBSTANCE: invention may be used for differential diagnostic of bronchial asthma (BA). Substance of the technique: a level of activity of effector caspase-3 of lymphocytes of peripheral venous blood is determined. If the level of activity of effector caspase-3 is no more than 0.98%, allergic BA is diagnosed, while the level of activity of effector caspase-3 is not less than 1.00%, non-allergic BA is diagnosed.

EFFECT: technique is described by simplicity, safety and adequate information value.

4 ex

FIELD: medicine.

SUBSTANCE: healthy children's blood serum is analysed for the enzymes aspartate aminotransferase (AST), glutamyl pyruvic transaminase (GPT), creatine phosphokinase (CPK), lactate dehydrogenase (LDG). If observing increasing activity of the enzyme AST and maintaining normal activity of the enzymes GPT, CPK, LDG, disturbed energy production of hepatocyte mitochondria in adolescents.

EFFECT: use of the technique enables high-accuracy diagnosing disturbed energy production in mitochondria in adolescents.

1 tbl

FIELD: medicine.

SUBSTANCE: there are offered a method and a kit for assessment of functional bypass activity of human complement component C9. The pharmaceutical preparation derinate is sorbed in microplate wells. Then the analysed sample containing the component C9 of unknown activity and porpoise complement is introduced in the analysed sample. It is followed with incubation, and after washing and drying of the dish, a conjugate of enzyme with component C9 antibodies and a substratum of this enzyme are added into the wells. The component C9 activity is evaluated by the amount of the prepared product of enzymatic reaction. The kit comprises the flat-bottomed microplate with sorbed derinate, conjugated enzyme and human complement component C9 antibodies, porpoise complement, substrate buffer and standard with known activity C9.

EFFECT: invention enables assessing the component C9 with using the preparation derinate as an classical activator of the complement.

2 cl, 1 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: bone isoenzyme of alkaline phosphatase and osteocalcin are evaluated on 22-24th weeks of pregnancy. If observing bone isoenzyme of alkaline phosphatase more than 6.0 Units/l and osteocalcin more than 9.0 ng/ml, developing postpartum osteopenic syndrome is predicted.

EFFECT: method enables the prenatal prediction of postpartum osteopenic syndrome.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method involves identifying in the 19th exon of the EGFR gene by polymerase chain reaction (PCR) with using upstream 5'-CTGTCATAGGGACTCTGGAT-3' and downstream 5'-CAGCAAAGCAGAAACTCACAT-3' primers that is followed by measuring a length of an amplified fragment by means of polyacrylamide gel electrophoresis. The L858R substitution in the 21st exon of the gene is detected by allel-specific PCR with using 5'-CACCCAGCAGTTTGGCCA-3', 5'-CACCCAGCAGTTTGGCCC-3' and 5'-GCATGAACTACTTGGAGGAC-3' primers. If observing in patients any of two types of mutations in the EGFR gene, the gefitinib therapy is prescribed.

EFFECT: invention provides high information value and reliability of tumour sensitivity test in the patients suffering lung cancer to gefitinib therapy with underlying simplicity and availability of the method.

2 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, oncology and haematology, and may be used for determining cardiotoxic complications in patients suffering chronic lymphatic leukemia and treated with polychemotherapy. Patient's blood serum is examined, and if observing no signs of infectious-inflammatory process and haemolysis, then blood is examined for lactate dehydrogenase by standartised UV test at temperature 37°C; the lactate dehydrogenase isoenzyme ratio is shown by enzyme electrophoresis, while enzyme linked immunosorbent assay provides calculating a tumour necrosis factor-α level. The examinations and assays run in with introducing chemopreparations and repeated in a week following the introduction and further throughout the whole polychemotherapeuty course in the same regiment, and in the end of the therapeutic course - once a month during the whole follow-up period. The lactate dehydrogenase level increasing by more than 510 IU/l and tumour necrosis factor-α increasing by more than 35 pg/ml, the lactate dehydrogenase-1 to lactate dehydrogenase-2 ratio increasing by more than 1 give evidence of potential development of cardiotoxicity of the chemopreparations in the patients, and subsequently, of life-threatening rhythm disturbances.

EFFECT: method enables well-timed, early determination of developing cardiotoxic complications of the chemotherapy in the given group of patients.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a subtype A human immunodeficiency viral strain, and can be used in virology, medicine and biotechnology. The presented type 1 IV742 human immunodeficiency viral strain is deposited in the State Collection of Viruses of Federal State Institution Ivanovsky Research Institution of Virology of the Ministry of Healthcare and Social Development of the Russian Federation, No. 1187. The strain possesses a stable reproductive activity. An infectious titre makes 6 lg 50% tissue cytopathic dose.

EFFECT: strain is a model for studying an antiviral activity of chemopreparations of new generation, as well as for creating vaccine preparations.

1 dwg, 3 tbl, 3 ex