RU2318022C2 - Medium for assay of hemolytic activity of culture of genus listeria - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a medium consisting of two layers - backing agar and covering agar. Backing agar comprises agar "Difko" and physiological solution. Covering agar comprises agar "Difko", yeast extract, dithiothreitol rabbit fibrin-free blood, sodium chloride, and beef-extract broth or Hottinger broth as a nutrient base. Invention provides study the listeriolysin expression level, to carry out total screening and differentiation of Listeria strains population based on feature of their hemolytic activity. Invention can be used in bacteriological diagnosis of listeriosis in research aims for characterization of strains by their biological activity.

EFFECT: valuable properties of medium.

3 tbl, 2 dwg, 3 ex

Description

The invention relates to veterinary and medical microbiology and can be used for bacteriological diagnosis of listeriosis, research purposes, characterization of strains by their biological activity.

In bacteriology, methods are widely used to determine the hemolytic activity of microorganisms in which this trait is associated with the manifestation of pathogenic properties, for example, such as pathogens of coccal infections, listeriosis, clostridiosis, hemophiles, colibacteriosis, yersineosis, mycobacteriosis, etc.

Three species are distinguished within the genus Listeria (L.monocytogenes, L.ivanovii, and L.seeligeri), which cause the lysis of human and animal erythrocytes (horses, cattle, sheep, rabbits) according to the beta type (I.A. Bakulov “Listeriosis agricultural animals ”, Moscow-1967, p.26-27). Listeria hemolysin is expressed during cultivation, both on liquid and on solid nutrient media, the rate and level of its production depending on the composition of the medium, incubation temperature and growth phase. A qualitative assessment of the hemolytic activity of Listeria is usually determined on classical blood agar, consisting of 1.5% meat-peptone agar with the addition of 5% defibrinated sheep blood. In this case, the studied daily broth culture of the microorganism is sown on the surface of blood agar. Crops are incubated at (36 ± 1) ° C for 24 ... 48 hours, after which the results are visually recorded by the presence of a narrow transparent zone of red blood cell lysis around the colonies or by the clarification of the medium under the colonies, indicating hemolysis, which is better seen when the latter are removed from the surface of agar (Listeriosis, Guidelines, No. 11, M. 2001).

The disadvantage of this environment is its low sensitivity. Significant agar density and a high concentration of red blood cells with a low production of listeriolisin make diffusion of exotoxins into the agar stratum difficult and, accordingly, the detection of a sign of hemolytic activity (Detection of Listeriolysin, the Thiol-Dependent Hemolysin in Listeria monocytogenes, Listeria ivanovii, and Listeria seisterigeri M. leisterteri M. leelteri M. leelteri leelteri M. leelteri L. and T. Chakraborty Vol. 57, No.8 // Infect, and Immun., Aug. 1989, p. 2350-2357). In this case, the accounting and evaluation of the sign is somewhat subjective. In addition, erythrocyte lysis under the colony may be caused by microbial cell metabolism products, i.e. to be non-specific.

To increase the sensitivity of detecting hemolytic activity in Listeria in microbiological practice, a test system based on the synergism with Rhodococcus eqvi and Staphylococcus aureus - CAMP test (GOST R 51921-2002 "Food products. Methods for the detection of bacteria Listeria monocytogenes") was proposed. The essence of the CAMP test is as follows: two-day cultures of hemolytic strains of Rhodococcus eqvi and Staphylococcus aureus are seeded on blood agar in two parallel vertical lines at a distance of 4 ... 5 cm from each other. Between the vertical culture lines of R.eqvi and S.aureus, the studied listeria cultures are inoculated with parallel horizontal lines at a distance of at least 1 cm from each other and 0.5 cm retreating from the vertical lines. Crops are incubated at (36 ± 1) ° С for 24 hours. Evaluation and accounting of the results is carried out by the presence of hemolysis (enlightenment of the medium) in the growth zone of the tested strains / isolates of listeria adjacent to the vertical lines of the cultures of R.eqvi and S.aureus.

Since the classic blood agar is used when setting up the CAMP test, it has the same drawbacks as described above. In addition, there is a need to maintain in laboratory conditions cultures of R.eqvi and S.aureus, which have a synergistic effect against hemolytic strains of Listeria, and these strains must be collection and not be subjected to repeated passivation in vitro, and L.monocytogenes strains are described in the literature. Synergism resistant (Cytolysins and the CAMP (synergistic hemolysis) phenomenon in Listeria. JAVazquez-Boland, C. Delgado, JFFemandez-Garayzabal, RITascon, M.Blanco, MBLopez, G. Suarez and L.Dominguez The Eleventh International Symposium on Problems of Listeriosis, 1992. - P.11-12).

The closest to the present invention is a single-layer blood agar with the addition of 5% defibrinated sheep blood, in which either Colombian (23.0 g / l peptone, 1.0 g / l corn starch, 5.0 g / l of sodium chloride, 15.0 g / l of agar-agar), or tryptone-soy agar (15 g / l of trypticase, 5 g / l of papain hydrolyzate of soy flour, 5.0 g / l of sodium chloride, 15.0 g / l agar-agar) as the most effective basis for testing the hemolytic activity of hemopositive microorganisms (Listeriosis, Methodical decree Nia №11, M. 2001)

Determination of hemolytic activity of listeria is as follows. The components of Colombian or tryptone soy agar are resuspended in the required amount of distilled water. The suspension is melted in a water bath, sterilized by autoclaving at 121 ° C for 15 minutes, and after cooling to 45 ° C, 5% v / v is introduced into it. defibrinated blood of a ram. Thus prepared blood agar is poured into 15 ... 20 ml in Petri dishes. After the agar layer has completely solidified, the studied daily broth cultures of Listeria are seeded on its surface. Crops are incubated at (36 ± 1) ° С for 24 ... 48 h, after which visual results are recorded by the presence of a narrow transparent zone of red blood cell lysis around the colonies or by clarification of the medium under the colonies. As can be seen from figure 1 on trypto-soybean and Colombian agar hemolysis zones (enlightenment of the medium under the colonies) are detected only in 1-3 and 1-2 of the five hemolytically active (1-5) strains of listeria, respectively (Table 1).

The use of Colombian or tryptone-soya agar as a basis ensures rapid and abundant growth of microorganisms, which are fastidious for the nutritional needs, and hemolytic reactions are more clearly manifested on these agars. In addition, corn starch serves as a source of energy and at the same time neutralizes toxic metabolites.

However, on blood agar, prepared as described above, only in strains of hyperproducers of hemolysins, a narrow zone of erythrocyte lysis is formed around the colonies. In the majority of hemolytically active strains of listeria, erythrocyte lysis manifests itself in the form of enlightenment of the medium under the colony, which makes accounting for the sign somewhat subjective. The disadvantage is due to the significant density of agar, which impedes the diffusion of hemolysin, as well as the fact that the number of hemolysin molecules produced by listeria cells at a concentration of red blood cells of 5% is not enough for their complete lysis in the hemolysis zone.

The aim of the invention is to develop an environment for determining the hemolytic activity of cultures of the genus Listeria, which is highly effective in detecting this trait, by creating better conditions for the synthesis and manifestation of the functional activity of thiol-dependent listeriolysin, as well as through the use of rabbit erythrocytes most sensitive to its action. The medium allows one to study the level of expression of listeriolysin, to conduct mass screening and differentiation of populations of strains / isolates of listeria on the basis of hemolytic activity.

Nutrient medium for determining the hemolytic activity of cultures of the genus Listeria, characterized in that it consists of two layers - epigastric agar and coating agar, while epigastric agar contains Difco agar and physiological saline, coating agar - Difco agar, yeast extract, dithiothreitol, defibrinated rabbit blood, sodium chloride, and as a nutrient base - meat and peptone broth or Hottinger broth, with the following components, wt.%:

- epigastric agar agar "Difco" - 1.0-1.5 saline - rest - cover agar agar "Difco" - 0.6-0.8 yeast extract - 0.9-1.1 dithiothreitol - 0,03 defibrinated rabbit blood - 3.0-3.5 sodium chloride - 0.35 meat and peptone broth, Hottinger Broth - rest

In relation to the prototype of the inventive medium has the following distinctive features.

The fundamental difference is that on the proposed medium, the growth of the microorganism culture occurs on the surface of a thin layer of coating agar, which is a nutrient medium that additionally contains yeast extract (DE) as a stimulator of cell growth of listeria and dithiothreitol (DTT) as an inducer of the synthesis and functional activity of a thiol-dependent listeriolysin. Reducing the agar content in the coating layer to 0.6 ... 0.8% contributes to better diffusion of hemolysins in the thickness of the agar with erythrocytes, and the use of more sensitive rabbit erythrocytes at a concentration of 3.0 ... 3.5% - the identification of their destructive activity . The use of a denser (1.0 ... 1.5%) transparent agar Difco as a substrate slows down the diffusion of listeriolysin directly under the colony and facilitates the detection of hemolysis due to the absence of a unmasking effect. In this regard, around the colonies of hemolytically active Listeria cultures, a transparent zone with lysed red blood cells is formed (Fig. 2), which, unlike the prototype, lends itself well to objective accounting and registration. As can be seen from figure 2, the erythrocyte lysis zone is observed not only under the colonies of hemolytically active strains, but around them (culture No. 1-5). Moreover, the erythrocyte lysis zone is recorded even in a culture of the Listeria seeligeri species (No. 5), which has the lowest hemolytic activity (Table 1).

Table 1 Comparative efficacy of detecting a sign of hemolytic activity in listeria No. p / p The name of the strain Hemolysis Colombian agar Tryptone Soy Agar Proposed environment one L.monocytogenes 634 - - + 2 L.monocytogenes 766 + + + 3 L.monocytogenes 9-72 + + + four L.monocytogenes 9-127 - + + 5 L.ivanovii + + + 6 L.seeligeri 100/100 - - + 7 L.murrayi - - - 8 L.welshimeri - - - 9 L.inocua - - -

The use of bilayer blood agar allows you to conduct dynamic observations, revealing the degree of enzymatic activity in any of the periods of development of the colonies, has high sensitivity and visibility.

The possibility of practical use of the invention is confirmed by examples of specific performance.

Example 1

To determine the presence of hemolytic activity of various types of listeria, 18 ... 24 h of broth culture is obtained.

Blood is taken from a rabbit by the standard method - puncture from the heart, following aseptic rules, into a vessel with beads to obtain defibrinated blood.

Preparation of coating agar is carried out as follows: to 50 ml of meat and peptone broth with a pH of 7.2 ... 7.4 (MPB - nutrient base) add a portion of 0.6 g (0.6 wt.%) Of Difco agar, 0, 9 g (0.9 wt.%) DE and 0.35 g (0.35 wt.%) Sodium chloride to obtain a final salt concentration in the mixture of 0.85% (taking into account its content in BCH) and the volume of the mixture is adjusted with meat and peptone broth up to 100 ml (98.15 wt.%). The mixture is melted in a water bath and autoclaved at 121 ° C for 15 minutes.

Two-layer blood agar is prepared: 1% Difco agar prepared in physiological saline is melted, cooled to 55 ... 60 ° C and poured into sterile Petri dishes of 15 ml each. To 96 ml (96 wt.%) Of coating agar, previously melted and cooled to 45 ° C, add 3.0 ml (3.0 wt.%) Of defibrinated rabbit blood, 1 ml (1.0 wt.%) 3% sterile solution of DTT, mix and then apply 5 ml of each to the cured layer of substrate agar.

table 2 Identification of a sign of hemolytic activity in listeria No. p / p The name of the strain Hemolysis BCH Environment BH based environment one L.monocytogenes 634 + + 2 L.monocytogenes 766 + + 3 L.monocytogenes 9-72 + + four L.monocytogenes 9-127 + + 5 L.ivanovii + + 6 L.seeligeri 100/100 + + 7 L.murrayi - - 8 L.welshimeri - - 9 L.inocua - -

The daily broth cultures of Listeria (L.monocytogenes, L.ivanovii, L.seeligeri, L.murrayi, L.welshimeri, L.inocua) are applied on plaques to the surface of two-layer blood agar. Crops are incubated at a temperature of 36 (± 1) ° C. The results are taken into account visually after 24, 36 and 48 hours. Around the isolated plaques L.monocytogenes, L.ivanovii and L.seeligeri, clearly defined completely transparent hemolysis zones are formed due to the complete destruction of red blood cells (Fig. 2, Table 2). There are no hemolysis zones around isolated plaques L.murrayi, L.welshimeri, L.inocua.

Example 2

To determine the presence of hemolytic activity of various types of listeria, their daily agar cultures are obtained and bilayer agar plates are prepared. The first layer in a Petri dish is poured into 20 ml of sterile molten 1.5% Difco agar prepared in physiological saline. Coating agar is prepared: 100 ml of Hottinger broth with a pH of 7.2 ... 7.4 (BH), 0.8 g (0.8 wt.%) Difco agar, 1.1 g (1.1 wt. .%) DE, 0.35 g (0.35 wt.%) NaCl to obtain a final salt concentration in the mixture of 0.85% (taking into account its content in BCh) is sterilized by autoclaving at 121 ° C for 15 minutes After cooling to 45 ° C, 95.5 ml (95.5 wt.%) Of coating agar, 3.5 ml (3.5 wt.%) Of defibrinated rabbit blood and 1 ml (1.0 wt.%) Are sterilely mixed. 3% sterile DTT solution. 5 ml of blanket blood agar is layered on the epigastric agar.

Daily surface agar cultures of Listeria (L.monocytogenes, L.ivanovii, L.seeligeri, L.murrayi, L.welshimeri, L.inocua) are applied to the surface of the prepared two-layer blood agar. Crops are incubated at a temperature of 36 (± 1) ° C. The results are taken into account visually after 24, 36 and 48 hours. Around the isolated colonies of L. monocytogenes, L. ivanovii and L. seeligeri, clearly defined completely transparent hemolysis zones are formed due to the complete destruction of red blood cells (Table 2). There are no hemolysis zones around isolated plaques L.murrayi, L.welshimeri, L.inocua.

Example 3

To determine the level of production of listeriolysin by various L.monocytogenes strains / isolates, the material of daily broth cultures of strains 766, 634, 9-72, 9-127 was plaque-coated onto the surface of a two-layer blood agar prepared as described above (examples 1 and 2).

Crops are incubated at a temperature of 36 (± 1) ° C. The results are taken into account visually after 36 hours by measuring the radius of the colony and the width of the hemolysis zone (clearly limited completely transparent zone due to the complete destruction of red blood cells) from the edge of the colony to the border of the hemolysis zone. The level of hemolytic activity is judged by the ratio of the radius of the colony (r _to ) and the width of the hemolysis zone:

r _to <the width of the hemolysis zone - tall r _k = or exceeds the width of the hemolysis zone in less than 1.5 times - average r _to > hemolysis zone width 1.5 times or more - low

The results of determining hemolytic activity are presented in tables 1 and 2.

Table 3 The results of determining the level of hemolytic activity of strains of L.monocytogenes No. p / p The name of the strain The radius of the colonies, mm The width of the hemolysis zone, mm Hemolytic activity level one L.monocytogenes 634 3.0 1,0 low 2 L.monocytogenes 766 4.0 1,5 low 3 L.monocytogenes 9-72 4.0 4.0 average four L.monocytogenes 9-127 3,5 4.0 tall

Claims (1)

Nutrient medium for determining the hemolytic activity of cultures of the genus Listeria, characterized in that it consists of two layers - epigastric agar and coating agar, while the epigastric agar contains Difco agar and physiological saline, coating agar - Difco agar, yeast extract, dithiothreitol, defibrinated rabbit blood, sodium chloride, and as a nutrient base meat-peptone broth or Hottinger broth with the following content of components in each layer, wt.%: Papillary agar: agar "Difco" 1.0-1.5 physical solution up to 100 Cover agar: agar "Difco" 0.6-0.8 yeast extract 0.9-1.1 dithiothreitol 0,03 defibrinated rabbit blood 3.0-3.5 sodium chloride 0.35 meat and peptone broth or hottinger broth up to 100

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