Method of determining sensitivity of microorganisms of salmonella genus to antibacterial agents. RU patent 2518372.
 Test-system for differentiating species and biotypes of bacteria of genus yersinia / 2518297Invention relates to field of microbiology and deals with test-system for differentiation of species and biotypes of bacteria of genus Yersinia. Claimed invention consists of a set of nutritional media, which contain substrates and reagents for determination of presence in microorganism of lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, urease, acetoin production, indole production, meliobiose fermentation, rhamnose fermentation, saccharose fermentation, sorbite fermentation, lipase, xylose fermentation, maltose fermentation, α-methyl-D-glucopyranoside fermentation, salicin fermentation, sorbose fermentation and raffinose fermentation. |
 Nutrient medium for extraction of bacteria of shigella kind from water objects / 2517750Nutrient medium is proposed for extraction of Shigella kind bacteria from water objects. The nutrient medium contains the following components: 0.1% alcohol solution of bromcresol green - 9.0 ml; 0.2% alcohol solution of methyl red - 3.0 ml; fermentative peptone - 0.5 g; extract of fodder yeast - 4.5 g; potassium dihydrophosphate - 8.7 g; caustic soda - 1.4 g; sodium chloride - 5.0 g; distilled water - up to 1000 ml; medium pH is 6.70-6.85. |
 Method of differentiating bacillus anthracis from other closely related species of genus bacillus based on determining differences in structure of chromosomal genes / 2514663Method includes sample preparation, DNA isolation, PCR statement. At that in carrying out PCR, the oligonucleotide primers are used, which are complementary to sequences of the chromosomal genes fliC and hom2, having the following sequences: fliC-F: 5'-TGGAGCAGTAACAATTGG-3', fliC-R: 5'-GCACCACTGATAGAAATGTTAG-3', hom2-F: 5'-GACGTGTTAAAAGAAGCCCA-3', hom2-R: 5'-CACCAATTTCGTCTTTTACA-3', followed by electrophoretic analysis of the amplification products, when the formation of the amplification product is 153 bps in size it is indicative of belonging of the strain under study to the species B.anthracis, formation of the amplification product with size of 550 bps is indicative of belonging of the strain under study to the other species of the genus Bacillus. |
 Differential diagnostic nutrient medium for identification of yersinia bacterium / 2511436Invention refers to biotechnology, microbiology, and concerns the recovery and identification of pseudotuberculosis and intestinal yersiniosis agents (Y. Pseudotuberculosis and Y. Enterocolytica). A nutrient medium contains microbiological agar (dry), lactose, glucose, urea, calcium chloride, 1% alcoholic phenol red, 1% alcoholic methylene blue and distilled water in specific proportions. |
| Method for preparing microgravimetric immunosensor / 2510830 Invention refers to biotechnology and microbiology. What is presented is a method for preparing a microgravimetric immunosensor. A surface of a quartz crystal resonator is activated first by plasma spray coating with polyethylene imine of molecular weight less than 10000 Da for 10 s in a vacuum unit at UHF field power 30 Wt. Thereafter, immunoglobulins from a solution with the protein concentration of 0.125 mg/ml are immobilised on the activated surface of the quartz crystal resonator. That is followed by washing in distilled water and drying in an air flow. |
 Method for quantitative assessment of bactericidal activity of disinfectants / 2510610Invention refers to microbiology and biotechnology. Analysed bacterial strains are inoculated on a dense nutrient medium. Paper disks impregnated with a disinfectant are applied. They are incubated in a temperature chamber until the bacteria start growing. The bacterial growth inhibition areas are measured. A quantity of the grown colonies is counted to construct a dependence diagram of the bacterial growth inhibition area and the quantity of the grown colonies after the disinfectant reaction. The diagram and Shughart inspection sheet are used to assess the disinfectant activity on specific types of the microorganisms. The disinfectants with mean measurements of the bacterial growth inhibition area are above an upper control limit of the Shughart inspection sheet are considered to be high bactericidal activity agents. The disinfectants with mean measurements of the bacterial growth inhibition area are below a lower control limit of the Shughart inspection sheet are considered to be low bactericidal activity agents. The disinfectants with mean measurements of the bacterial growth inhibition area between the control limits of the Shughart inspection sheet are considered to be mean bactericidal activity agents in relation to all analysed agents. |
 Method of species identification of l.casei/paracasei, l.fermentum, l.plantarum and l.rhamnosus / 2508406Invention refers to a method of species identification of L.casei/paracasei, L.fermentum, L.plantarum, L.rhamnosus lactobacilli. The proposed method involves performance of a PNR reaction with species-specific primers; besides, primers are built, which are specific to the first gene of operon FIFO ATP of synthase (a gene of subunit a) and a gene of uracylphosphoribosyltransferase, which precedes it, for L.casei/paracasei and L.rhamnosus and a gene of uracyltransport protein for L.plantarum and L. fermentum. |
 Dry chromogenic feed medium for detection of coliform bacteria and e.coli (versions) / 2508400Invention can be used for detection of coliform bacteria and E.coli in specimens of food products and water at performance of bacteriological tests. Feed medium includes a nitrogen source represented by meat peptone or pancreatic hydrolysate of fish flour, sodium chloride, dibasic sodium phosphate, potassium monophosphate, sodium pyruvate, L-tryptophane, sodium dodecyl sulphate, 6-chloro-3-indolyl-β-D-galactopyranoside (Salmon - GAL), 5-bromine-4-chloro-3-indolyl-β-D-glucoronide-(X-GLUC), isopropyl- β-D1-tiogalactopyranoside (IPTG) and microbiological agar in the specified ratio. |
| Dry differential diagnostics feed medium for detection and consideration of e.coli and coliform bacteria / 2508399 Invention can be used for detection and considering of E.coli and coliform bacteria in water, food products, clinic material, etc. Feed medium contains pancreatic hydrolysate of fish flour dried with Tergitol 7 on the bases of 0.1 g of Tergitol 7 per 6 g of dry pancreatic hydrolysate of fish flour, yeast extract, 1-water D (+) lactose, bromthymol blue, sodium dodecyl sulphate, 2,3,5-triphenyltetrazolium chloride, sodium carbonate and microbiological agar in the specified ratio. |
| Method of identifying vibrio bacteria / 2506313 Invention relates to microbiology and biotechnology. Material to be investigated - pure culture of rod-like, gram-negative, glucose-fermenting, oxidase-positive or oxidase-negaive bacteria - is collected first. The investigated daily bacterial culture is seeded on the surface of nonselective nutrient agar (GRM-agar) with 1% sodium chloride. A paper disc is then placed seeded surface, said disc containing vibriostatic substance niclosamide (2,5-dichloro-4-nitrosalicylanilide) in amount of 10 mcg or 16 mcg per disc. The seeded material is incubated in aerobic conditions at 35°C for 24 hours. Vibrio bacteria are indicated a zone of inhibited bacterial growth around the disc. |
 Strain of rhodococcus sp-destructor of petroleum hydrocarbons / 2518349Strain of Rhodococcus sp. is deposited in the All-Russian Collection of Microorganisms under the registration number Ac-2046 D. The strain exhibits high destructive activity against petroleum hydrocarbons included in the composition of oil slurries, as well as against crude oil and black oil fuel. |
| Two-phase nutritional medium for thin layer cultivation of helicobacter pylori and method of its realisation / 2518304 Invention relates to field of microbiology and can be applied in medicine. Method includes preliminary inoculation of analysed material on Columbia agar with addition of 5% of sheep blood. Thermostatting is performed under microaerophilic conditions in CO2 incubator at 37°C for 3-5 days at higher humidity. Sampling colonies and their re-inoculation on Petri dishes with chocolate agar are performed and thermostatting is carried out under microaerophilic conditions in CO2 incubator at 37°C for 3-5 days at higher humidity. When thermostatting stage is completed Petri dishes with chocolate agar are poured on the top with liquid nutritional medium based on Schaedler broth, enriched with 10% cattle serum with their further thermostatting in CO2 incubator at 37°C for 72 hours. |
| Test-system for differentiating species and biotypes of bacteria of genus yersinia / 2518297 Invention relates to field of microbiology and deals with test-system for differentiation of species and biotypes of bacteria of genus Yersinia. Claimed invention consists of a set of nutritional media, which contain substrates and reagents for determination of presence in microorganism of lysine decarboxylase, ornithine decarboxylase, tryptophan deaminase, urease, acetoin production, indole production, meliobiose fermentation, rhamnose fermentation, saccharose fermentation, sorbite fermentation, lipase, xylose fermentation, maltose fermentation, α-methyl-D-glucopyranoside fermentation, salicin fermentation, sorbose fermentation and raffinose fermentation. |
 Anti-obesity preparation, anti-obesity food product or drink, preparation improving tolerance to glucose and food product or drink for improving tolerance to glucose / 2518293Group of inventions relates to field of biotechnology. Strain Bifidobacterium breve MCC 1274 FERM BP-11175 demonstrates low coefficient of conversion of linoleic acid into conjugated linoleic acid. Strain demonstrates coefficient of conversion of linoleic acid into conjugated linoleic acid not higher than 10%. To reduce or prevent obesity or to improve tolerance to glucose said strain is introduced in efficient quantity to subject requiring it. Also claimed are food product and drink, which contain preparation based on said strain. |
| Nutrient medium for submerged cultivation of tularemia microbe / 2518282 Nutrient medium for submerged cultivation of tularemia microbe contains as a base, that provides the content of amino nitrogen in the medium of not less than 0.32%, dry enzymatic hydrolyzate of fibrin obtained from waste of whey-vaccine production, and the components - glucose and calcium pantothenate at a predetermined ratio. |
| Nutrient medium for extraction of bacteria of shigella kind from water objects / 2517750 Nutrient medium is proposed for extraction of Shigella kind bacteria from water objects. The nutrient medium contains the following components: 0.1% alcohol solution of bromcresol green - 9.0 ml; 0.2% alcohol solution of methyl red - 3.0 ml; fermentative peptone - 0.5 g; extract of fodder yeast - 4.5 g; potassium dihydrophosphate - 8.7 g; caustic soda - 1.4 g; sodium chloride - 5.0 g; distilled water - up to 1000 ml; medium pH is 6.70-6.85. |
 Method to prepare medicated product from live strains and microorganisms of lactobacilli and bifidobacteria "lb-complex plus" / 2517734Method provides for cultivation at 37±1°C of strains of lactobacilli and bifidobacteria in the medium and packing of liquid product with account of daily dose necessary for patients. The medium contains components in the following quantities: caseine hydrolysate dissolved by distilled water, 0.33-0.4 g/l, sodium chloride 5 g/l, fructose 10 g/l, peptone 2 g/l, agar-agar 0.75 g/l for lactobacilli, for bifidobacteria - 1.0 g/l, ascorbic acid 0.25 g/l, distilled water 0.67-0.6 g/l. Strains-producers are Lactobacillus plantarum 8 RA-3, Lactobacillus fermentum 39, Lactobacillus fermentum 90 TC-4, Bifidobacterium bifidum 791, Bifidobacterium longum 379, Bifidobacterium bifidum 1. For lactobacilli and bifidobacteria they prepare accordingly media with different content of agar-agar, amine nitrogen in caseine hydrolysate 160-170 and 180-200 mg% and medium pH 7.8-8.0 and 8.5-8.6. Starting from the first generation the bifidobacteria and lactobacilli are cultivated for 24±1 hours, two strains together, one separately. The produced biomass is mixed with the fresh nutrient medium at the ratio of 1:1000, and strains of lactobacilli are cultivated 24±1 hours, strains of bifidobacteria - 48±1 hours. Upon completion of cultivation of biomass of strains they are mixed at the ratio of 2:1:2:1. |
 Nourishing composition, including probiotics and improving sleep pattern / 2517616Invention relates to application of probiotic bacterial strain for production of a probiotic composition for reducing sleep disorders and/or improvement of sleep quality in people and animals. As a bacterial strain used is Lactobacillus reuteri DSM 17938 or Bifidobacterium longum NCC 3001 (ATCC BAA-999). |
| Strain of fungus stagonospora cirsii davis having herbicidal activity against canada thistle / 2515899 Strain of the fungus Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 has herbicidal activity against canada thistle and species closely related to it - S. Cirsii. It was deposited in the State Collection of Microorganisms of All-Union Research Institute of Plant Protection under the collection number of Stagonospora cirsii Davis of All-Union Research Institute of Plant Protection 1.42 and can be used for biological control of canada thistle. |
 Medication (versions), composition (versions) and application of medication (versions) for reduction of halitosis / 2515113Invention relates to versions of medication for reduction of halitosis, versions of compositions based on said medications and versions of application of said medications. Device for reduction of halitosis represents strains of microorganisms Lactobacillus acidophilus, selected from group Lactobacillus acidophilus DSM 19825, Lactobacillus acidophilus DSM 19826 and Lactobacillus acidophilus DSM 19827. Version of said medication represents culture supernatant of said strains. Also claimed are compositions, which contain said microorganisms, and their application for reduction of halitosis. |
 Strain of lactobacilli lactobacillus paracasei cncm i-2116 (ncc 2461) eliciting ability to prevent intestine colonization with pathogenic microorganisms causing diarrhea, supernatant of its culture and oral agent for prophylaxis and/or treatment of diarrhea-associated disorders / 2243779Invention relates to the strain Lactobacillus paracasei CNCM I-2116 used for diarrhea prophylaxis causing by pathogenic microorganisms. Supernatant of this strain culture elicits ability to prevent colonization of intestine with pathogenic microorganisms causing diarrhea also and this strain is designated for preparing agent used for prophylaxis and/or treatment of disorders associated with diarrhea. Agent for oral administration represents therapeutically effective dose of the strain L. paracasei CNCM I-2116 or supernatant of its culture and acceptable foodstuff. Invention provides the enhanced viability of the strain in its applying and effectiveness in prophylaxis of adhesion to intestine cells and invasion to intestine cells of pathogenic microorganisms causing diarrhea. |
FIELD: biotechnology.
SUBSTANCE: test kind of microorganisms of the genus Salmonella is incubated, which is taken at a final concentration of approximately 1000 cells/ml, with the test antibacterial agent in the known concentration previously specified for each antibacterial agent, which is optimal for exposure to the specified kind of microorganisms. Incubation is carried out at a temperature of 37°C for 24 hours. The samples are added to the aptamers specific to test species of living microorganisms, fluorescently labelled, at a final concentration of 100 nM. It is incubated for the second time for 20 minutes. The sample is examined using flow cytometry. The level of fluorescence of aptamers is determined by graphs made with a flow cytometer using a program which enables to display a percentage value. The part of bound and un-bound bacteria with the aptamers fluorescently labelled is determined. The amount of the living or non-living organisms in the sample is determined.
EFFECT: method enables to determine with the high degree of accuracy the sensitivity of microorganisms to antibacterial agents.
2 dwg, 1 ex
The invention relates to the definition of antibacterial resistance of the genus Salmonella using aptamers.
Currently, a growing number of highly pathogenic strains of pathogens of infectious diseases, the treatment efficiency of which depends on the resistance of bacteria to used in the treatment of antibiotics.
Infections caused by resistant organisms, often fatal and hamper the fight against infectious diseases, as patients remain carriers of the disease over a long period of time that allows the transmission of resistant strains to other people. In connection with constant increase of drug resistance of bacteria medicine needs fast, high precision methods of determining the degree of resistance of microorganisms to antibiotics.
Today, the sensitivity of microorganisms to antibiotics define the following known methods.
How to serial dilutions of the direct quantitative determination of the main indicator characterizing the activity of antibacterial drug, i.e. the magnitude of his minimum overwhelming concentration [susceptibility of microorganisms to antibiotics (guidelines MUK 4.2.1890-04). Clinical Microbiology and antimicrobial chemotherapy. 2004, V.6, №4].
This method can be accomplished with the use of liquid culture media by serial dilution antimicrobial drugs twice, receiving the number of samples is equal to the number of dilutions. Microorganisms are incubated with the received samples in equal concentrations approximately 10 6 CFU/ml, then the result is evaluated visually by turbidity samples (either through an in the case of analysis of immunological tablet). How to serial dilutions can be done by seeding microorganisms on Petri dishes with agar containing a serial dual cultivation of antibiotics. However, the result can only be cleaning validation of culture. In addition, time requires preparation Petri dishes, seeding them microorganisms cultivation and counting of colonies.
Famous disco-diffusion method, based on the ability of antibacterial preparations to diffuse of impregnated paper disks in a nutrient medium with the inhibition of the growth of microorganisms, have been sown on the surface of the agar (to determine the sensitivity of microorganisms to antibiotics (guidelines MUK 4.2.1890-04). Clinical Microbiology and antimicrobial chemotherapy. 2004, V.6, №4].
For the implementation of disco-diffusion method requires a special preparation of a Petri dish as the thickness of the medium must have a certain value, and the surface on which produce the pouring of molten agar in the cups should be strictly horizontal. In addition, to implement the process only requires standardized quality disks impregnated antibacterial drugs, the production of which in laboratory conditions is impossible.
In addition to the known ways and developed on their basis of standard methods to determine the sensitivity of microorganisms to antibiotics also known methods described in the applications and patents for inventions.
The known method of determination of antibiotic resistance in microorganisms, including the identification of microorganisms (WO/2009/095258, C12Q 1/04; G01N 33/58, 08.06.2009). The method includes the manufacture of labeled antibiotic, incubation labeled antibiotic with tested microorganisms in the conditions that allow microorganisms to contact the antibiotic, and detection labeled antibiotic microorganisms. Labeled antibiotic also incubated with organisms of the same species, sensitive to this antibiotic. The number of Antibiotika in the tested organisms compared with the number of antibiotic sensitive microorganisms. Through this, determine the sensitivity of microorganisms to antimicrobial drug.
The described method requires additional time expenses for manufacturing labeled antibiotic and incubation with sensitive to the microorganism, in addition incubation with tested microorganisms. And also requires the availability of additional sensitive to this antibiotic organisms of the same species.
The known method of determination of antibiotic profile bacteria and treatment of the patient therapeutically effective antibiotics (WO/2009/137139, C12Q 1/68; C12N 1/20, 11.12.2009). Method of determination of antibiotic resistance includes: receiving of nucleic acid bacteria, visualization nucleic acid bacteria, obtaining restriction map, nucleic acids, comparison restriction map nucleic acid database restriction maps and determination of antibiotic resistance of bacteria by matching the specified regions nucleic acids with relevant regions in the specified database. The described method is rather labor-intensive, as it includes nucleic acid bacteria.
Also known "Method of determining the sensitivity of microorganisms to antimicrobial agents" (RU 2321855, G01N 33/48, G01N 21/00, 12.10.2006), namely, that on the samples of various antimicrobial drugs act laser radiation by measuring the intensity of the fluorescence of samples in different time intervals. On trial without antimicrobial effect of laser radiation in the same intervals and measure the intensity of the fluorescence of a sample without antimicrobial agents. Then carry out a comparison of fluorescence intensity of samples with antimicrobial drugs, normalized by the corresponding intensity of fluorescence of a sample without antimicrobial agents. When reducing the normalized intensity of the fluorescence of samples with antimicrobial drugs by a certain amount diagnose inhibitory effect of antimicrobials in microorganisms.
This method of determining the sensitivity of microorganisms is time consuming and takes a lot of time, because it involves the irradiation of samples laser in various successive periods of time, re-measurement of the intensity of fluorescence in comparison with samples not containing antimicrobial drugs.
The famous "Method of determining the sensitivity of microorganisms multidrug resistant to the combinations of antibacterial drugs" (RU 2388827, C12Q 1/04, C12N 1/00, 10.05.2010)adopted for the prototype consists in that sowing of studied cultures of microorganisms carried out in a test tube with mesopetalum broth, and then make impregnated antibacterial drug standard paper disks and incubated for 12-18 hours at a temperature of 36 of + / -1 C. The sensitivity of microorganisms to the combination of antibacterial drugs visually assess the transparency of the contents of the test tube. No turbidity says about the sensitivity of microorganisms to the combination of antibacterial drugs.
This method of determination of antibiotic sensitivity, despite the reduction of labor costs in comparison with standard methods to identify, very accurate, as it may not provide quantitative estimates. And requires the special standard disks impregnated with antibacterial drugs.
The present invention is to increase the accuracy of the method of determining the sensitivity of microorganisms of the genus Salmonella to antibacterial drugs.
The problem is solved by a method of determining the sensitivity of microorganisms of the genus Salmonella to antibiotics, including incubation check microorganisms species studied with antibacterial drug, according to the invention, antibacterial drug known previously established for each antibiotic concentration, for optimal exposure for a given type of microorganisms, incubated with microorganisms of a certain type, taken at a final concentration of about 1000 cells/ml, with a temperature of 37 C for 24 hours, after incubating the samples add aptamers labeled with fluorescent label, in final concentration 100 nm specific to the audited species of living organisms, and he incubated for 20 minutes, the sample is examined using flow cytometry, the level of fluorescence of aptamers, determined on the charts, built with the flow cytometer using a program that allows you to display in percentage values, showing the proportion of bound peroxidase or not bound peroxidase bacteria aptamers labeled with fluorescent label, determine the number in the sample of living or non-living microorganisms.
In the invention describes how to determine the sensitivity of microorganisms of the genus Salmonella to antibiotics through aptamers, can specifically bind to live microorganisms and not to contact non-living (i.e. subjected to the action of antibacterial preparations). How fundamentally different the fact that for the determination of antibiotic sensitivity of microorganisms used aptamers.
The technical result of the invention is achieved through the implementation of the opportunity to obtain a quantitative assessment of living and non-living microorganisms in the level of fluorescence of aptamers, and thus improve the accuracy of determination of antibiotic resistance of bacteria to one or another antimicrobial drug. Furthermore, the method excludes the human factor in the form of visual estimation of the sensitivity of bacteria on the turbidity of the sample.
Aptamers - synthetic single-stranded molecules of RNA or DNA, obtained with the help of technology SELEX and capable specific binding with virtually any biological target. Aptamers are of high sensitivity and specificity and at a lower cost, as are synthesized chemically.
In microorganisms, sensitive to the effects of the antibiotic changing the structure of the cell wall, while resistant to antibacterial drug organisms retain their viability, and the structure of their cell walls is not changed. Therefore, when exposed to a microorganism antibacterial drugs, you can determine their resistance to antibiotics through analysis of svyazyvaet with aptamers that are specific to this type of living organisms.
The method is illustrated by the graphs in figure 1 shows a graph of determination of antibiotic sensitivity Salmonella Enteritidis to three antibacterial drugs; figure 2 shows a graph of determination of antibiotic sensitivity Salmonella Typhimurium to three antibiotics.
For the implementation of the method using the following reagents:
1. Pool DNA aptamer (or DNA aptamer), selected to the specified type of living organisms, negative targets for the selection of which served as non-living organisms of a given species.
2. Liquid nutrient medium.
3. Bidistilled water.
4. Phosphate buffer with Ca 2+ and Mg 2+.
5. Organisms of a given species.
6. Antibiotic sensitivity of microorganisms to which you want to define.
The method is as follows.
Antibacterial product (if it is in solid or powder form) is first dissolved in the water, and then in a nutrient medium. Then add the specified type of microorganisms in the amount of approximately 1,000 cells/ml The final concentration of antibiotic should be optimal for the influence on the given type of microorganisms (the optimal concentration is usually specified in the annotations to this antibacterial preparation).
The sample is incubated in thermostat at the temperature of 37 C for 24 hours.
Temperature of 37 C, selected for the approximation of the experiment to the real conditions of antibacterial preparations on bacteria in the body. The incubation period of 24 hours due to the possibility to evaluate the effectiveness of antibacterial drug on pathogenic microorganisms.
After incubation to add samples fluorescently labeled aptamers that are specific to this type of microorganisms R. Salmonella, and incubated for 20 minutes.
The sample is examined using flow cytometry.
The level of fluorescence of aptamers, who contacted with bacteria, spoke about the number located in a sample of living or non-living organisms, and thus their sensitivity to check the antibiotic. The level of fluorescence to determine the graphs constructed using a flow cytometer using a program that allows you to display in percentage values indicate the proportion of bound peroxidase or not bound peroxidase bacteria aptamers labeled with fluorescent label. See the graphs were obtained in the processing of data obtained in the analysis of samples using a flow cytometer FC-500 (Beckman Coulter Inc., USA) using the Kaluza 1.1. Graphics were built by the program automatically.
The following is an example of the method of determining the sensitivity of microorganisms to antibiotics, particularly to antibiotics.
Example. Determined sensitivity of Salmonella Typhimurium and Salmonella Enteritidis to the following antibiotics: "Flemoxin of Solutab, Azithromycin and Cefotaxime". To determine the sensitivity used the following reagents:
1. DNA aptamer labeled with a fluorescent tag specific to Salmonella Typhimurium, negative target for breeding which served as inanimate Salmonella Typhimurium, and bacteria of some other species. DNA aptamer labeled with a fluorescent tag specific to Salmonella Enteritidis, negative target for breeding which served as inanimate Salmonella Enteritidis, and bacteria of some other species.
2. Liquid nutrient medium prepared using the powder Mueller-Hinton.
3. Phosphate buffer with CA 2+ and Mg 2+.
4. Bidistilled water for dilution antimicrobial drugs.
Each of the studied antibacterial drugs "Flemoxin of Solutab, Azithromycin and Cefotaxime" were diluted with bidistilled water and then in liquid nutrient medium, in final concentration, featured in the annotations to these antibiotics. Then on Wednesday made Salmonella Typhimurium and Salmonella Enteritidis, in final concentration of about 1000 cells/ml In the control group consisted live Salmonella Typhimurium and Salmonella Enteritidis, mixed with a liquid nutrient medium. And Salmonella Typhimurium and Salmonella Enteritidis exposed to the temperature of 95 degrees C for 15 minutes.
They received a total of 10 samples:
1. Nutrient medium + "Cefotaxime" + Salmonella Typhimurium
2. Nutrient medium + "Cefotaxime" + Salmonella Enteritidis
3. Nutrient medium + Azithromycin" + Salmonella Typhimurium
4. Nutrient medium + Azithromycin" + Salmonella Enteritidis
5. Nutrient medium + "Flemoxin of Solutab" + Salmonella Typhimurium
6. Nutrient medium + "Flemoxin of Solutab" + Salmonella Enteritidis
7. Nutrient medium + Salmonella Typhimurium after 15 min temperature of 95 degrees C
8. Nutrient medium + Salmonella Enteritidis after 15 min temperature of 95 degrees C
9. Nutrient medium + Salmonella Typhimurium
10. Nutrient medium + Salmonella Enteritidis
All samples were incubated in a CO2 incubator for 24 hours at a temperature of 37 C, after which the samples were diluted to get the number of bacteria approximately 1,000 cells/ml After that added aptamers labeled with fluorescent label in final concentration 100 nm, and incubated for about 30 minutes. Then the samples were centrifuged at 5000 rpm for 10 minutes, shot adosados, and added to the precipitate phosphate buffer with CA 2+ and Mg 2+ . The obtained samples were investigated on a flow cytometer FC-500 (Beckman Coulter Inc., USA). For data processing software has been used Kaluza 1.1.
The results obtained using flow cytometry, reflected in figure 1 and 2.
Figure 1 shows the determination of antibiotic sensitivity Salmonella Enteritidis using aptamers.
In the analysis linking aptamers with samples containing Salmonella Enteritidis daily after incubation with "Flemoxin", "Cefotaxime and Azithromycin", fluorescence was observed, which testifies to the absence in the sample live bacteria, and therefore, the sensitivity of Salmonella Enteritidis to data antibiotics (figure 1 curves 1, 2, 3). Aptamers, proskurovskii with viable Salmonella Enteritidis exposed to the temperature of 95 degrees C for 15 minutes, not associated with bacteria (figure 1, curve 5). This confirms that aptamers not have affinity to the non-living bacteria.
Fluorescence Salmonella Enteritidis related aptamers, characteristic only for bacteria, not treated with antibacterial drugs (curve 4), with the number of living bacteria is 39% of the total number, that, apparently, is connected with insufficient stability of this type of bacteria to certain conditions of the experiment.
This complete absence of fluorestsentsii daily after incubation of samples with "Flemoxin", "Cefotaxime and Azithromycin confirms that Salmonella Enteritidis has a high sensitivity to the selected antibacterial drugs in the recommended concentration.
Figure 2 shows the determination of antibiotic sensitivity of Salmonella Typhimurium with aptamers.
From the above figure 2 graphs, it follows that the fluorescence of Salmonella Typhimurium related aptamers, characteristic for bacteria, not treated with antibacterial drugs (curve under the 9 rooms), with the number of living bacteria, as the results generated by the program, is 97,4%, and for bacteria treated Flemoxin (curve 6) and Cefotaxime (curve 7), remain alive respectively 10,8 and 12.0% of microorganisms. In the samples, proskurovskikh with Azithromycin, fluorescence was observed, that is, live bacteria in them. Thus necrost of aptamers to non-living bacteria and their specificity to live, as well as experience with Salmonella Enteritidis, confirms the fact of absence of fluorescence in the incubation aptamers with Salmonella Typhimurium subjected fifteen minutes to temperatures of 95 degrees C (figure 2 curve 10).
Thus, Salmonella Typhimurium insufficiently sensitive to the drugs Flemoxin and Cefotaxime in the recommended known doses, while there is a 100% death of bacteria at influence on them of Azithromycin in established for this drug, the dose.
Thus, during the analysis of antibiotic sensitivity was found that Salmonella Enteritidis has sensitivity to any of the studied antibacterial drugs "Flemoxin of Solutab, Azithromycin and Cefotaxime in the established for them concentrations for optimal impact on this type of microorganism. Salmonella Typhimurium is more resistant to drugs "Flemoxin of Solutab" and "competitive" and has sensitivity to the drug Azithromycin".
The use of aptamers in the present method allows a high degree of accuracy to determine the sensitivity of microorganisms R. Salmonella to antibacterial drugs that are currently used in the treatment of the diseases caused by these organisms, and, in turn, will choose to treat infections of a number of drugs are those drugs that pathogens are more sensitive, that is to accelerate the healing process of the patient.