RU2416635C2 - Chromogenic culture medium for detecting and identifying klebsiella - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: culture medium contains dry nutrient agar, glucose, 5- aminosalicylic acid, an extract of nutrient yeast, paraaminobenzoic acid, bromthymol blue, tris-buffer, sodium carbonate, brilliant green and microbiological agar.

EFFECT: invention shortens duration of identifying klebsiell.

3 ex

Description

The invention relates to medicine, namely to sanitary and clinical microbiology, and can be used in the study of various biological material, as well as environmental objects with suspected Klebsiellosis.

It is known that over the past decades in all economically developed countries of the world, significant changes in the structure of human infectious pathology have been noted: against the background of a decrease in the incidence rate of a number of classical infections, an increase in diseases caused by opportunistic microorganisms is recorded, among which one of the leading places belongs to Klebsiella ( 1, 3, 5, 6, 11, 12). They also describe the detection of Klebsiella in the environment: in water, food products, on the surface of instruments in medical facilities, clothing of medical staff, etc.

A fairly wide distribution of Klebsiella in the environment is known, in connection with which their importance both as indicator microorganisms and as potentially pathogenic, causing pathology in humans, increases.

Klebsiella can cause nosocomial infections with a severe course of the disease, often fatal, they are highly resistant to disinfectants, as well as antibacterial drugs. The latter complicates the treatment and reduces its effectiveness (1, 3, 5, 10).

The largest role in human pathology among microorganisms of the genus Klebsiella is played by K. pneumoniae, K.oxytoca, K.mobilis (1, 11).

Due to the lack of clinical features of the disease caused by Klebsiella, a correct and timely diagnosis of Klebsiella can be made only on the basis of bacteriological diagnosis (5, 12).

Nutrient media for the isolation of Klebsiella are known - these are the usual differential diagnostic nutritional traces in which the principle of lactose fermentation is laid (Endo, Levin, Ploskirev, McConkey medium). However, on these media it is impossible to distinguish Klebsiella from other bacteria of the Escherichia coli group (BHEC), because they all break down lactose (8, 12).

A known medium for the isolation of Klebsiella containing the antibiotic carbenicillin as a selective agent (4) that inhibits concomitant microflora. However, most enterobacteria are weakly sensitive to this antibiotic, as a result of which its selective properties are insufficient to suppress the growth of all BGPs, creating favorable conditions for the growth of only Klebsiella. And the sucrose contained in the medium, split by all BGKP, does not make it possible to distinguish them from each other.

Another medium, based on the principle of inhibition of concomitant microflora, is Boyko OV et al. (2). It contains meat-peptone agar, lactose, and safranin T. The latter, being an oxidation-reduction indicator, does not completely suppress the associated microflora, which decompose lactose and grow in colonies of different sizes, but the same color as Klebsiella, which makes differentiation difficult.

Media are known for the isolation of Klebsiella by the sign of inositol cleavage (3, 9). But these media support the growth of not only K. pneumoniae, but also Serratia spp: both types of microorganisms grow in the form of medium and large, nucleated, smooth yellow colonies. Differentiation of Klebsiella from serrations according to colony morphology is very difficult (3).

Therefore, to determine the affiliation of the selected cultures to Klebsiella, it is necessary to set up a number of confirmatory tests, and therefore, considerable time and material costs (3, 5). These are tests for motility, hydrogen sulfide, urea, phenylalanine deaminase, Simmons citrate, malonate, lysine decarboxylase, inositol. The total cost of time from 3 to 5 days.

At the same time, when conducting microbiological studies, an important condition is the speed of obtaining results, which contributes to early diagnosis, timely conduct of therapeutic and anti-epidemiological measures.

The closest solution to the problem of the same purpose in terms of the set of characteristics and the achieved effect is a nutrient medium for the isolation of Klebsiella (7). The principle of this medium is based on fermentation by Klebsiella inositol. The composition of the medium is in g / l:

Casein Yeast Hydrolyzate 19.7 Mesoinositis 7.0 Indicator bromothymol blue 0.5 2,4-dinitrophenol 0.001 Potassium sulfate 1,0 Magnesium sulfate 1,0 Microbiological agar 1,0

The reason that impedes the achievement of the technical result indicated below when using a dry nutrient medium for the isolation of Klebsiella, adopted as a prototype, is the ability to cleave inositol by other representatives of the Enterobacteriaceae family, namely Serratia marcescens, Yersiniae enterocolitica, Proteus rettgeri, Providencia (12), which grow by this medium is difficult to distinguish by color from Klebsiella colonies, which does not provide a clear differentiation between them (see table).

The purpose of the invention is to obtain a nutrient medium for the isolation and identification of Klebsiella based on the identification of their genus-specific enzyme - 5-aminosalicylate decarboxylase.

To achieve the specified technical result when implementing the invention, dry nutrient agar containing amine nitrogen — 2.5 ± 0.5%, chlorides — 18 ± 2%, jelly strength, was introduced into the medium as a nutritional basis for maintaining the growth of Klebsiella and other UPEs. g - 300 ± 35, residual humidity not more than 7%, pH of the medium - 7.3 ± 0.2, regardless of its modification and the origin of the raw material. To identify the unique intracellular genus-specific enzyme of Klebsiella - 5-aminosalicylate decarboxylase, which no other member of the Enterobacteriaceae family produces, 5-aminosalicylic acid (5-ASA) is introduced into the medium, which cleaves under the influence of 5-aminosalicyl decarboxylase of brown color to form the product staining only Klebsiella colonies and the environment around them in brown (13, 14).

For microorganisms that do not produce 5-aminosalicylate decarboxylase, glucose and a bromothymol blue indicator are introduced into the medium, which allow the formation of acid from glucose during the growth of these microorganisms that turn yellow in color, in contrast to brown Klebsiella colonies. In order to compensate for the inhibitory effect of 5-ASA on Klebsiella, bacterial growth stimulants, fodder yeast extract (ECD) and para-aminobenzoic acid (PABA), were introduced on Wednesday. And also the medium contains distilled water as a necessary element for the preparation of a nutrient medium intended for the cultivation of microorganisms.

The identification of Klebsiella on the proposed medium is carried out simultaneously with their selection, i.e. in one step. Additional identification tests are not required, which reduces the time to establish a diagnosis to 24 hours instead of several days necessary to confirm or exclude that the selected inositol-positive culture belongs to the Klebsiella genus when using the medium taken as a prototype. Gram-positive microorganisms are suppressed when sowing from a dilution of 10 ^-1 as a result of the introduction of brilliant green into the medium.

The composition of the proposed environment Daghrom Klebsiella agar in g / l distilled water:

Nutrient agar, dry 35.0-40 Glucose GOST 6038-79 5.0-6.0 5-aminosalicylic acid from Sigma 2.0-3.0 Fodder yeast extract FS42-3441-97 3.0-5.0 Para-aminobenzoic acid, Sigma 0.01-0.02 Bromothymol blue TU 6-09-5430-90 0.08-0.09 Tris buffer TU6-09-4292-76 1.0-1.5 Sodium carbonate GOST 5100-73 0.6-0.7 Diamond green, zd chemical reagent. Shostka 0.0001-0.0002 Microbiological agar GOST 17206-84 2.5-3.0

This composition of the medium provides a clear differentiation of Klebsiella (brown) in color from other BGKP and proteins (yellow), which is not possible when using traditional lactose-containing media (Endo, Levine, etc.) or media with inositol.

A culture medium for the isolation and accelerated identification of Klebsiella - DagHrom Klebsiella agar is prepared as follows.

Example 1. In 1 liter of distilled water, 35.0 g of dry nutrient agar, 3.0 g of feed yeast extract, 0.01 g of para-aminobenzoic acid, 0.08 g of bromothymol blue, 2.5 g of microbiological agar are successively added to 5.0 g of glucose, 2.0 g of 5-aminosalicylic acid, 1.0 g of Tris buffer, 0.6 g of sodium carbonate and 0.0001 g of brilliant green. The mixture is thoroughly mixed, boiled three times for 1-2 minutes with stirring, avoiding agar burning, until a coarse bubble foam appears. The medium is cooled to a temperature of 45-50 ° C, and then poured into sterile Petri dishes. After the agar plate has hardened, the plates with the medium are dried in an thermostat at 37 ° C for 50-60 min in compliance with aseptic rules. Ready-to-use medium is transparent, bottle-green, pH 7.4 ± 0.2. The test material is applied to the surface of the medium and rubbed with a sterile spatula. Incubate inoculation at a temperature of 37 ° C for 24 hours.

Example 2. It differs from example 1 in that 37.0 g of dry nutrient agar, 4.0 g of feed yeast extract, 0.015 g of para-aminobenzoic acid, 0.085 g of bromothymol blue, 2.7 g of microbiological agar are sequentially added to 1 liter of distilled water. 5.5 g of glucose, 2.5 g of 5-aminosalicylic acid, 1.25 g of Tris buffer, 0.65 g of sodium carbonate and 0.00015 g of brilliant green. Next - as in example 1.

Example 3. It differs from example 1.2 in that 40 g of dry nutrient agar, 5.0 g of extract of feed yeast, 0.02 g of para-aminobenzoic acid, 0.09 g of bromothymol blue, 3.0 are sequentially added to 1 liter of distilled water. g of microbiological agar, 6.0 g of glucose, 3.0 g of 5-aminosalicylic acid, 1.5 g of Tris buffer, 0.7 g of sodium carbonate and 0.0002 g of brilliant green. Further, according to example 1, 2.

The proposed environment supports the growth of all BGCs. After 24 hours of incubation of the crops (at 37 ± 1 ° C), typical brown colonies with a diameter of 1.5 to 2.0 mm in S-form are formed on the proposed medium K.pneumoniae, K.oxytoca, and K..mobilis. At the same time, brown precipitate is clearly visible around the Klebsiella colonies. Colonies of other enterobacteria - Escherichia, cytrobacter, enterobacter, serrations, protea, etc. grow in the form of yellow colonies typical of each species against the background of a bottle-green color of the nutrient medium. There is no brown precipitate around these colonies (see table).

Using the proposed environment can significantly reduce the time of identification of Klebsiella and reduce material costs.

Bibliographic data

1. Bondarenko V.M., Barkus M.M. Enterotoxic ability of the strains of the genera Klebsiella and Enterobacter isolated in acute intestinal diseases in children. ZhMEI, 1986, 7, p. 28-32.

2. Boyko O.V., Nikolaev A.A., Boyko A.V., Lutsky D.A. Nutrient medium for the isolation of Klebsiella. Bulletin "Inventions, utility models", M., 2003, No. 34, part 2, p. 522.

3. Gorchenina L.V., Kiseleva B.S. Klebsiella isolation media. ZhMEI, 1985, 1, p. 19-22.

4. Ibragimov F.Kh. and other Selective nutrient medium for the allocation of Klebsiella BIPM, 2004, No. 30, 1 hour, p.140.

5. Krasnogolovets V.N., Kiseleva B.S. Klebsillosis infections. M., 1996, pp. 9-39, 47, 63-64, 69-70, 79, 114.

6. Marie P.R., Shay I.R. Clinical Microbiology. Quick start guide. Per. from English M., 2006, p. 52-72, 386.

7. Mejidov M.M. Handbook of microbiological culture media. M., 2003, p. 63-64.

8. General and sanitary microbiology with the technique of microbiological research. Edited by A.S. Labinskaya, L.P. Blinkova, A.S. Yeshchina, M., 2004., 525, 535-546.

9. Sandra F.D. et al. (USA). Culture medium for the isolation of Klebsiell / Enterobacter. Patent No. 708998. BIPM, 1980, No. 1, p. 260.

10. Sarkulova M.N. Microbiological characteristics of pathogens of nosocomial infections in urological patients. ZhMEI, 2005, 5, pp. 101-103.

11. Sidorenko S.V., Rezvan S.P., Eremina L.V. et al. Etiology of severe hospital infections in the intensive care unit and antibiotic resistance among their pathogens. G. Antibiotics and chemotherapy. 2005, 2-3, vol. 50, p. 33-41.

12. Enterobacteria. A guide for doctors. Ed. V.I. Pokrovsky. M., 1985, p. 171-175, 181-193, 186.

13. Ellens, D.J. and Gielkens, A.L.J. J. Immunol. Meth. 37, 325 (1980).

14. "Sigma". Catalog of reagents for biochemistry and research in the field of natural sciences. 2002-2003, p. 157.

COMPARATIVE CHARACTERISTICS OF THE DIFFERENTIATING PROPERTIES OF THE PROPOSED ENVIRONMENT AND THE KNOWN, TAKEN FOR THE PROTOTYPE.

Test - strains Famous environment Proposed environment Inositol Colony color 5-aminosalicylic acid Colony color 1. K. pneumoniae 3534/51 + yellow + Brown precipitate 2. K. oxytoca + yellow + Brown precipitate 3. K. mobilis + yellow + Brown precipitate 4. S. marcescens 1 + yellow - yellow 5. P. rettgori + yellow - yellow 6. Y. enterocolitica + yellow - yellow 7. E.coli Su 3912/41 O55 K: 59 - blue - yellow 8. E. cloacae 10005 - blue - yellow 9.S. freundii 101/57 - blue - yellow 10. S. aureus 25923 no growth no growth no growth no growth

Claims (1)

Chromogenic nutrient medium for the detection and identification of Klebsiella, containing a nitrogen source, 5-aminosalicylic acid, bromothymol blue, microbiological and distilled water agar, characterized in that it additionally contains glucose, para-aminobenzoic acid, extract of fodder yeast, Tris-buffer, sodium carbonate, brilliant green, and as a source of nitrogen - dry nutrient agar in the following ratio of components, g / l of distilled water:
nutrient agar, dry 35.0-40 glucose 5.0-6.0 5-aminosalicylic acid 2.0-3.0 yeast extract 3.0-5.0 para-aminobenzoic acid 0.01-0.02 bromothymol blue 0.08-0.09 tris buffer 1.0-1.5 sodium carbonate 0.6-0.7 diamond green 0.0001-0.0002 microbiological agar 2.5-3.0