RU2099417C1 - Pyrazineamide-containing nutrient medium for determination of medicinal resistance of tuberculosis pathogen and their identification - Google Patents

Abstract

FIELD: medicinal microbiology. SUBSTANCE: nutrient medium has the following components, g: potassium dihydrogen phosphate 4.0-9.0; magnesium sulfate 0.2-0.6; sodium citrate 0.3-0.9; ferric ammonium sulfate 0.01-0.04; ferrous ammonium sulfate 0.01-0.04; aminoacetic acid 0.5-1.3; α-aminosuccinic acid 0.3-0.9; succinic acid 0.8-1.6; ammonium succinate 1.2-2.4; sodium dihydrogen phosphate 1.3-2.4; brilliant green 0.01-0.04, and also, ml: glycerol 7.0-17.0; hydrolysin 15-50; homogenized egg mass 200-500, and also: penicillin 100000-300000 U/mcl; pyrazineamide 50-200 mcg/ml, and water - up to 1 l. EFFECT: enhanced effectiveness of medium. 1 tbl

Description

 The invention relates to the field of medical microbiology and can be used in the laboratories of the research institute, the system of TB facilities, as well as veterinary medicine.

 The characteristic of the analogue. Mediums are known for determining drug resistance to pyrazanamide. Most often, Middlebrook 7H10 and Levenshtein-Jensen media containing pyrazanamide are used to determine drug resistance. However, there are no analogues of the proposed proposal (as a complex). In the scientific and information-patent literature, as well as in the practice of phthisiobacteriology, the substrate and the method are equally well known both for determining drug resistance to pyrazanamide and for identifying microbacteria.

 Criticism of the prototype. Due to the fact that resistance to pyrazinamide is detected in a strictly defined pH range (5.0 to 5.5), the above media must be acidified to the indicated pH values. However, with acidification, the growth properties of the media deteriorate. So when subculturing a suspension containing a standard inoculum on a dense Levenshtein-Jensen medium with a neutral pH (6.8 7.2), the result is read on the 14th day. Growth in a medium acidified to 5.0 5.5 slows down, and the result can be read only at the 4th 6th week. In addition, the disadvantage of the prototype is a complex composition, including expensive and scarce imported ingredients. Additional disadvantages of the low pH Levenshtein-Jensen medium are ruptures of the sloping surface of the medium that occur during coagulation, and extremely late dates for obtaining the results of a pyrazinamide resistance study (up to 6 weeks) in relation to individual clinical cases with chronic tuberculosis (4). An additional disadvantage of the medium is the need to grow mycobacteria in a gaseous medium containing carbon dioxide.

 The essence of this proposal. The purpose of the proposed facility is: a) the creation of a new substrate with pyrazinamide based on domestic available chemotherapeutic agents and drugs; b) the exclusion of an additional technological stage of acidification of the environment; c) ensuring the growth of the tested cultures of mycobacteria in a short time; d) cheaper analysis.

The solution of the tasks is provided by a specially designed substrate, which includes the following components, g per 1 liter of the finished medium:
Potassium phosphate monosubstituted 4.0 9.0
Magnesium sulfate 0.2 0.6
Sodium citrate 0.3 0.9
Ammonium iron (III) sulfate 0.01 0.04
Ammonium iron (II) sulfate 0.01 0.04
Aminoacetic acid 0.5 1.3
α-amino succinic acid 0.3 0.9
Succinic acid 0.8 1.6
Ammonium succinic acid 1.2 2.4
Monosubstituted sodium phosphate 1.3 2.4
Diamond Green 0.01 0.04
Glycerin, ml 7.0 17.0
Hydrolysin, ml 15.0 50.0
Homogenized egg mass, ml 200 500
Penicillin 100000 300000
Water Else
Final pH of the medium 5.4 5.6
Before coagulation, pyrazinamide is introduced into the medium to provide a concentration of 25-50-75-100-200-400 μg / ml.

As can be seen from the component composition, the proposed substrate consists of available domestic preparations, and the pH achieved without subtraction provides those unique conditions under which pyrazinamide acts most effectively. In addition, specially selected new chemical compounds have a dual function: they contribute to the formation of low pH and at the same time are stimulators of the growth of altered mycobacteria. A special mode of compaction and coagulation of the media provides the required consistency and promotes the rapid growth of mycobacteria. In addition, the proposed method allows incubation of mycobacteria under ordinary conditions, without the use of a CO ₂ incubator.

 The study of resistance to pyrazanamide in mycobacteria isolated from patients showed that the proposed method allows to give the result to the clinic on the 10th 14th day.

 The minimum inhibitory concentration of pyrazinamide for most laboratory strains: Academy, H37RV, Erdman is 25 μg / ml. Sensitive strains should be considered those that do not grow on the proposed medium with 50 to 100 μg / ml pyrazinamide. In addition to determining resistance to pyrazinamide, the proposed method allows to establish the type of mycobacteria (human or bovine). The differentiating ability of media with pyrazinamide is shown in table 1.

Example 1. In distilled water heated to 80 90 ^o C, successively dissolved, g: potassium phosphate monosubstituted 4.0; magnesium sulfate 0.2; sodium citrate 0.3; ammonium iron (II) sulfate 0.01; ammonium iron (III) sulfate 0.01; aminoacetic acid 0.5; a-amino succinic acid 0.3; succinic acid 0.8; ammonium succinic 1,2; monosubstituted sodium phosphate 1.2; diamond green 0.01; glycerin 7 ml; hydrolysin 15 ml; homogenized egg mass 200 ml, distilled water the rest. The salt components are dissolved in the indicated solution, autoclaved at 1 atm (121 ^° C) for 25 minutes, cooled to room temperature, homogenized egg mass, brilliant green solution, 100,000 units are added. penicillin, pyrazinamide to obtain working concentrations of 25-50-75-100-200-400 μg / ml. Part of the medium without pyrazinamide is used as a control. Coagulate at 78 ^o C for 30 minutes The sterility of media with and without pyrazinamide is tested by incubation at 37 ^° C for 48 hours. Sowing with a density of 10 million microbial cells in 1 ml is inoculated with 0.2 ml for each test tube and control. Indication of growth is made visually, starting from the 8th day daily.

Example 2. In distilled water heated to 80–90 ^° C., successively dissolved, g: potassium phosphate monosubstituted 9.0, magnesium sulfate 0.6; sodium citrate 0.9; ammonium iron (II) sulfate 0.04; ammonium iron (III) sulfate 0.04; aminoacetic acid 1.3; a-amino succinic acid 0.9; succinic acid 1.6; ammonium succinic acid 2.4; monosubstituted sodium phosphate 2.4; diamond green 0.04; glycerin 17 ml; hydrolysin 50 ml; homogenized egg mass of 500 ml; penicillin 300,000 units distilled water the rest. The salt components are dissolved in this order, autoclaved at 1 atm (121 ^o C) for 25 min, cooled to room temperature, add homogenized egg mass, a solution of brilliant green, 300,000 units. penicillin, pyrazinamide to obtain working concentrations of 25-50-75-100-200-400 μg / ml. Further, as in example 1.

Example 3. In distilled water heated to 80 ^° -90 ^° C., successively dissolved, g: monosubstituted potassium phosphate 6.5; magnesium sulfate 0.4; sodium citrate 0.6; Ammonium iron (II) sulfate 0.025; ammonium iron (III) sulfate 0.025; aminoacetic acid 0.9; a-amino succinic acid 0.6; succinic acid 1.2; ammonium succinic acid 1.8; monosubstituted sodium phosphate 1.8; diamond green 0.025; glycerin 12 ml; hydrolysin 32 ml; homogenized egg mass 350 ml; penicillin 200,000 units Before coagulation, pyrazinamide is introduced into the medium to provide concentrations of 25-50-75-100-200-400 μg / ml. Further, as in example 1.

 Feasibility or other efficiency. The proposed environment consists of affordable non-deficient ingredients of domestic production. According to the source of amine nitrogen, the proposed medium is 75 times cheaper than the Levenshtein-Jensen medium. The proposed medium has a semi-dense consistency, and therefore the consumption of the sealant (dietary eggs) per unit volume is 2 times less than in the Levenshtein-Jensen medium. In general, 1 ton of the proposed medium is cheaper than 1 ton of the Levenshtein-Jensen medium by approximately 1000 rubles.

In addition, significant savings in working time are provided by a one-stage technology for preparing the medium. Using the proposed medium does not require special conditions (CO ₂ incubator), provides rapid growth of test dies in the “acidic” pH range (5.4 to 5.6), and serves as a reliable test for typing mycobacteria.

Sources of information
1. ZN Kantos, IE Amadio, EA Catalini, MDC Zonardo, Z. Dominguez, D. Cuffo, A. Zaslo, O. Zatini, I. Poggio, H. Reyes Iutierres, MT Vacenzuela. Pyrasinamidase activity and susceptibility to pyrazinamide in clinical isolates of Mycobacterium tuberculosis. Pyrazinamide suseptibility of M. tuberculosis.

 2. Slottmeier K.D. Blat R.F. and Kubica I.P. Pyrazinamide suseptibility and amidase activity of tubercle bacilli, American Review of Respiratory Desease 1967, 97, 1072.

 3. Mycobacterial culture collection US Goverment Printing Office 1980.

 4. Auth. St. USSR N 1666530, C 12 N 1/20, C 12 Q 1/04, 1991.

Claims (1)

Nutrient medium with pyrazinamide for determining the drug resistance of tuberculosis pathogens and their identification, containing potassium phosphate monosubstituted, magnesium sulfate, brilliant green, glycerin, homogenized egg mass and water, characterized in that it additionally contains sodium citrate, iron (III) ammonium sulfate iron (II) ammonium sulfate, aminoacetic acid, α-aminosuccinic acid, succinic acid, ammonium succinic acid, monosubstituted sodium phosphate, hydrolysis in, penicillin, pyrazinamide with the following quantitative ratio of components:
Potassium phosphate monosubstituted, g 4.0 9.0
Magnesium sulfate, g 0.2 0.6
Sodium citrate, g 0.3 0.9
Ammonium iron (III) sulfate, g 0.01 0.04
Ammonium iron (II) sulfate, g 0.01 0.04
Aminoacetic acid 0.5 1.3 g
α-amino succinic acid, g 0.3 0.9
Succinic acid, g 0.8 1.6
Ammonium succinic acid, g 1.2 2.4
Monosubstituted sodium phosphate, g 1.3 2.4
Diamond green, g 0.01 0.04
Glycerin, ml 7.0 17.0
Hydrolysin, ml 15.0 50.0
Homogenized egg mass, ml 200 500
Pyrazinamide, μg / ml 50 200
Penicillin, u / μl 100,000 300,000
Water, l Up to 1o

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