RU2715329C1 - Nutrient medium for separation and identification of non-fermenting bacteria - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to medical microbiology. Nutrient medium for separation and identification of non-fermenting bacteria contains nutrient broth dry, extract of fodder yeast for microbiological nutrient media, D-glucose, D-galactose, sodium chloride, sodium thiosulphate, iron (III) citric acid hydrate, sodium carbonate, sodium sulphate, phenol red, bromothymol blue, calcium carbonate, microbiological agar and distilled water at preset amounts of components.

EFFECT: invention allows to differentiate non-fermenting bacteria from fermenting with simultaneous primary differentiation of different bacteria according to color change of medium and / or colony color.

1 cl, 3 tbl, 3 ex

Description

The invention relates to the field of medical microbiology and can be used to isolate and identify non-fermenting gram-negative bacteria (NFB) from clinical material: separable wounds, sputum, urine, feces, with suspected contamination of normal microflora.

Non-fermenting gram-negative bacteria are one of the main causative agents of nosocomial infections (NDS). The incidence of nosocomial infections caused by the NSE reaches 15% of all nosocomial infections associated with aerobic and facultative aerobic gram-negative bacteria. At the same time, their species composition has expanded significantly in recent years.

Isolation of NFB strains on simple nutrient media such as meat-peptone agar or blood is complicated, since cultures of Staphylococcus and other concomitant bacteria masking the presence of NFB grow more strongly in these media, while the identification of NFB on these media is also difficult due to the low enzymatic activity of these strains and the relative proximity of their morphological and cultural characters.

To isolate NPF strains, such as Pseudomonas aeruginosa, from clinical samples and to differentiate them from other pseudomonads based on the formation of the pyocyanin pigment, BD Pseudomonas Isolation Agar (agar for the isolation of pseudomonads. Agar contains Bacto peptone (a source of carbon and nitrogen), an antimicrobial agent irgasan (Irgasan), selectively inhibiting concomitant gram-positive and gram-negative bacteria, glycerin as an energy source, contributing to the production of the pigment pyocyanin, as well as magnesium chloride and potassium sulfate, that form the blue or blue-green pigment of pyocyanin from P. aeruginosa. This pigment diffuses into the environment surrounding the growth zone (Instructions for use - ready-to-use environment in cups RA-257002.04 Rev .: April 2013 https://www.bd. com / resource.aspx? IDX = 25359)

To isolate and identify NPFs, a new generation of differential diagnostic media is also used - fluorogenic and chromogenic, which allow identification of various microorganisms directly in the process of cultivation on nutrient media at the initial inoculation stage. The principle of operation of these media is based on the identification of highly specific enzymes in the desired microorganisms. The composition of these media includes a chromogenic substrate - a substance, upon cleavage of which by enzymes specific for a certain type of microorganism, colored and / or fluorescent products are formed. As a result, colonies of the desired microorganisms and / or medium are stained in a certain color, or acquire the ability to fluorescence under ultraviolet radiation.

Known HiFluoro agar base for Pseudomonas aeruginosa - HiFluoro Pseudomonas Agar Base (HiMedia Laboratories Pvt. Limited, India), designed for the selective isolation and identification of P. aeruginosa bacteria from clinical material using a fluorescence method, which includes cetrimide to inhibit the growth of concomitant microorganisms mixture. P. aeruginosa, when grown on a medium, destroys the fluorogenic substrate, releasing fluorogen, which gives visible fluorescence under ultraviolet radiation (Instructions for use: https://art-medika.com/catalog/mikrobiologia/chromo/product-487.html (http: //www.himedialabs.ru/m1469).

Known chromogenic culture media designed to isolate non-fermentative bacteria Burkholderia cepacia from clinical samples:

- BD Cepacia Medium (Becton Dickinson GmbH Heidelberg / Germany), containing nitrogen sources (peptones, ammonium sulfate), phosphates to maintain a stable pH, a carbon source (sodium pyruvate), NPF growth factors (magnesium, iron), and inhibitors to suppress concomitant microflora (bile salts, crystalline violet, ticarcillin and polymyxin B), and, as an indicator of pH, phenol red. During the metabolism of sodium pyruvate, sodium ions accumulate, causing an increase in pH, which leads to a color change from yellow-orange to pink or red around B. cepacia colonies and intense pink in areas of dense growth (BD Cepacia Medium BD OFPBL Agar Instructions for application - ready-to-use medium in cups RA-254481.04 Ed .: Oct 2014: https://www.bd.com/resource.aspx?IDX=25339):

- BD OFPBL Agar (Becton Dickinson GmbH Heidelberg / Germany), contains inhibitors of concomitant bacteria (bacitracin - an inhibitor of gram-positive microorganisms and Neisseria, polymyxin B - gram-negative microflora), potassium hydrogen phosphate to maintain a stable pH, and bromothymol blue as an indicator of pH. When lactose is fermented into acidic products, and, accordingly, lowering the pH of the medium, the pH indicator bromothymol blue changes the color of the medium from blue to yellow, B. cepacia colonies will also have a yellow color. At the same time, the Instructions for the use of the medium contain restrictions on the use of the medium, namely that growth of other microorganisms, for example B. gladioli, which are similar in appearance to B. cepacia (yellow colonies), is possible on OFPBL agar, and it is emphasized that this medium should not used as the sole medium for identification of B. cepacia. (BD Cepacia Medium BD OFPBL Agar Instructions for use - ready-to-use medium in cups RA-254481.04 Rev .: Oct 2014: www.bd.com/resource.aspx?IDX=25919).

Thus, well-known foreign media designed to isolate certain types of NSFs do not provide the possibility of simultaneous isolation and identification of several types of NSFs, especially when conducting large-scale epidemiological monitoring of associations of strains of NSFs circulating inside hospitals, are also expensive.

Also known is the domestic differential diagnostic medium for the isolation of non-fermenting gram-negative bacteria (SU 1351975), which contains a selective agent, 2,3,5-triphenyl-tetrazolium chloride and, as an indicator, bromothymol blue, and allows you to isolate NFB from clinical material and differentiate them from sugar fermenting enterobacteria and proteins.

The specified environment has the following composition:

This medium, being chromogenic, provides the color of the group of non-fermenting bacteria in burgundy, fermenting - in yellow, Proteus mirabilis forms black colonies. But this medium does not provide differentiation between individual representatives of non-fermenting bacteria.

The aim of the invention is a differential diagnostic environment that allows the differentiation of non-fermenting bacteria from fermenting and, at the same time, the primary differentiation of different representatives of non-fermenting bacteria by changing the color of the medium and / or bacterial colonies, and containing domestic ingredients.

The task is achieved by introducing into the nutrient medium a two-component indicator system (phenol red + bromothymol blue), which ensures the differentiation of non-fermenting bacteria from fermenting and, at the same time, the primary differentiation of different representatives of the NFB according to the change in colony color and the nature of growth. The addition of calcium carbonate prevents excessive acidification of the environment with the waste products of bacteria. The medium contains domestic ingredients.

The proposed nutrient medium (ModSI - Modified Medium with Indicator) has the following composition:

The method of preparation of the nutrient medium ModSI. For the preparation of 1 liter of the sample medium (dry nutrient broth, fodder yeast extract for microbiological nutrient media, D-glucose, D-galactose, sodium chloride, sodium sulfate, iron (III) citric acid, sodium carbonate, sodium sulfate, phenol red, bromothymol blue, calcium carbonate, microbiological agar) is dissolved in a small amount of distilled water, the volume is adjusted to 1 liter, heated to boiling until the ingredients are completely dissolved; The pH of the medium is adjusted to 7.5 ± 0.1. ModSI medium is poured into vials and sterilized at 0.5 atm. within 30 minutes. The finished medium is lilac-pink, opaque. Before use, the bottle with the medium is melted in a boiling water bath and poured into sterile Petri dishes of 15-20 ml. Ready cups with ModSI medium are stored at a temperature of +6 ° C for a week.

Example 1. Selection of indicator concentrations in the composition of the Modsi medium The proposed Modsi nutrient medium was prepared according to the above method in 2 variants differing in the concentration of indicators: phenol red and bromothymol blue when added to the Modsi medium, they were used in two concentrations: 0.025 g and 0 each. 05 g

Option 1

Вариант 2

Option 2

The following test strains were used to determine the optimal concentrations of indicators in the medium: NSE - Pseudomonas aeruginosa No. 453, Burkholderia cepacia No. B-7518, Stenotrophomonas maltophilia No. B-7520. These test strains are grown on slanted meat-peptone agar (MPA) for 24 hours at + 37 ° C, the grown cultures are washed off with a sterile isotonic sodium chloride solution (0.85%), a suspension of bacteria is prepared according to the optical standard of turbidity 10 ME and titrated to the content 1000 mt / ml. 0.1 ml of the obtained suspension of crops is sown with a continuous lawn with a spatula of Drigalski for 2 variants of the proposed ModSI medium. Crops are incubated at + 37 ° C for 24 hours. The bacterial growth efficiency and the differentiating properties of the 2 variants of ModsI are determined by the number of colony forming cells (CFU) and by the change in colony color and the nature of growth.

The results are presented in table 1.

As can be seen from table 1, the optimal concentrations in the composition of the proposed nutrient medium ModSI indicators phenol red - 0.05 g and bromothymol blue - 0.05 g (option 2), at which the growth efficiency of non-fermenting bacteria is 91-97 CFU, and differentiating the properties of the medium provide for the difference between different representatives of non-fermenting bacteria among themselves, in contrast to the medium in option 1 with an indicator concentration of 0.025 g.

Example 2. The growth efficiency of bacteria (NPF, gram-negative enterobacteria, staphylococci) on the proposed environment Modsi (in comparison with the differential diagnostic environment according to SU 1351975, nutrient medium Endo and yolk-salt agar).

When studying the efficiency of bacterial growth, the following test strains were used: NPF - Pseudomonas aeruginosa No. 453, Burkholderia cepacia No. B-7518, Stenotrophomonas maltophilia No. B-7520; gram-negative enterobacteria - Escherichia coli M-17, Proteus vulgaris No. 869, Proteus mirabilis No. 878, Klebsiella pneumoniae No. 63, Salmonella typhimurium No. 67, Salmonella enteritidis No. 2269, Salmonella dublin No. 1976; gram-positive cocci - Staphylococcus aureus No. 209-p, Staphylococcus epidermidis No. 136.

These test strains are grown on slanted meat-peptone agar (MPA) for 24 hours at + 37 ° C, the grown cultures are washed off with a sterile isotonic sodium chloride solution (0.85%), a suspension of bacteria is prepared according to the optical standard of turbidity of 10 ME, and titrated to the content of 1000 MT / ml. 0.1 ml of the obtained suspension of cultures is sown with a continuous lawn with a Drigalski trowel: on the proposed ModSI medium - all test strains (NFB, gram-negative enterobacteria, staphylococci); on the differential diagnostic environment according to SU 1351975 - test strains of the NSE, gram-negative enterobacteria; Endo - test strains of gram-negative enterobacteria; on yolk-salt agar - test strains of staphylococci. Crops are incubated at + 37 ° C for 24 hours. Take into account the number of colony forming cells (CFU), and the percentage of plating on plates with medium, based on the number of microbial bodies in 1 ml of microbial suspension.

The results are presented in table 2.

As can be seen from table 2, on the proposed nutrient medium ModSI, the growth efficiency of non-fermenting bacteria was 91-97 CFU (91-97%), which is comparable with the results of the growth of NFB on a differential diagnostic medium according to SU 1351975 (91-100 CFU).

At the same time, a rather high growth efficiency was established on the proposed medium of gram-negative enterobacteria (CFU 91-98) and staphylococci (CFU 90-92), corresponding to the growth rates of these bacteria on selective, nutrient media (Endo medium for gram-negative enterobacteria, ZhSA - staphylococci), which will allow the use of ModSI for growing a wide range of microorganisms.

Example 3 The study of the differentiating properties of the proposed nutrient medium Modsi and control environments for non-fermenting bacteria, gram-negative enterobacteria, staphylococci

In studying the differentiating properties of ModsI, the following test strains were used: NSE - Pseudomonas aeruginosa No. 453, Burkholderia cepacia No. B-7518, Stenotrophomonas maltophilia No. B-7520; gram-negative enterobacteria - Escherichia coli M-17, Proteus vulgaris No. 869, Proteus mirabilis No. 878, Klebsiella pneumoniae No. 63, Salmonella typhimurium No. 67, Salmonella enteritidis No. 2269, Salmonella dublin No. 1976; gram-positive cocci - Staphylococcus aureus No. 209-p, Staphylococcus epidermidis No. 136. These test strains are grown, a suspension is prepared, and plated on test media plates as described in Example 2.

Data on the study of differentiating properties are presented in table 3.

As can be seen from table 2, on the proposed nutrient medium ModSI, NFB test strains (P.aeruginosa, B.cepacia, S.maltophilia) differed in colony color and growth pattern both from test strains of other bacteria (gram-negative enterobacteria, staphylococci), so and among themselves.

Thus, from the above tables it follows that, on the proposed ModSI medium, the growth efficiency of all microorganisms taken as test strains is quite high and comparable with the media intended for isolation and differentiation of the corresponding microorganisms (Endo medium for gram-negative enterobacteria, ZhSA for staphylococci , differential diagnostic environment for the isolation of non-fermenting gram-negative bacteria according to SU 1351975), which indicates the possibility of using ModSI for growing shir Who spectrum of microorganisms.

When evaluating the differentiating properties of the ModSI medium, it was found that the proposed medium allows us to distinguish the NFB test strains (P.aeruginosa, B.cepacia, S.maltophilia) as test strains of other bacteria (gram-negative enterobacteria, staphylococci) by the color of the colonies and the nature of growth. so among themselves.

Thus, the proposed nutrient medium ModSI can be used to differentiate non-fermenting bacteria from fermenting and, at the same time, primary differentiation of different representatives of non-fermenting bacteria, by changing the color of the medium and the different color of the colonies, during the initial inoculation of samples of clinical material, and, which is especially important, when epidemiological monitoring of NSF strains circulating in the hospital.

Claims (2)

Nutrient medium for isolation and identification of non-fermenting bacteria, containing dry nutrient broth, fodder yeast extract for microbiological culture media, D-glucose; D-galactose, sodium chloride, sodium sulfate, ferric (III) citric acid, sodium carbonate, sodium sulfate, calcium carbonate, microbiological agar, distilled water, in which the phenol red and bromothymol blue indicators are additionally added with the following amounts of components: dry broth 20.0 g microbiological feed yeast extract            1.0 g nutrient media D-glucose                                                                                  1.0 g D-galactose                                                                                     20.0 g sodium chloride                                                                                 5.0 g sodium sulfate                                              0.3 g iron (III) citrate water                                             0.6 g sodium carbonate                                                                      0.5 g sodium sulfite                                                         0.5 g phenol red                                                                      0.05 g bromothymol blue                                                                 0.05 g calcium carbonate                                                                    5.0 g microbiological agar                                                      11.0 ± 2.0 g distilled water                                                             up to 1 l pH                                                                                               7.5 ± 0.1