RU2296163C2 - Method for evaluating specific activity of bacteriophages - Google Patents

Abstract

FIELD: analytical methods in microbiology.

SUBSTANCE: substance of the method resides in performing, under human embryo cell culture monolayer conditions, common 2-h culturing of test strains of microorganisms with suitable therapeutical bacteriophage or multifunctional (polyvalent) bacteriophage, which includes a phage corresponding to test culture. Specific activity of bacteriophage is evaluated from degree of adhesion of test strain of microorganism to monolayer cells compared to control (cell monolayer and test strain without bacteriophage).

EFFECT: speeded up determination and enabled evaluation of therapeutical bacteriophage due to approaching evaluation conditions to microorganism conditions.

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Description

The invention relates to the field of medicine, medical toxicology, microbiology, biology when testing various bacteriophages (for example, therapeutic), namely, to assess the specific activity of therapeutic bacteriophages.

The essence of the method boils down to the fact that under standard conditions a monolayer of the culture of human embryo cells, the test strain of the microorganism is co-cultured with the corresponding therapeutic bacteriophage or combined (multivalent) bacteriophage, which includes the phage corresponding to the used test culture of the microorganism.

Currently, bacteriophages are widely used to identify isolated microorganisms, as well as a therapeutic agent. A quick response is necessary in the clinic for an early diagnosis and the use of rational therapy, especially since the preparations of a therapeutic bacteriophage are used only after determining the sensitivity of the culture isolated from the patient to the phage.

Bacteriophage preparations are various dosage forms (dry tablet, liquid purified concentrate, liniment) that are active against the most common types of staphylococcus, protea, streptococcus, E. coli and Pseudomonas aeruginosa. As an example of a polyvalent bacteriophage, the official preparation of the pyobacteriophage combined purified concentrated in the form of a purified liquid concentrate was used.

Tests to determine the sensitivity of microorganisms to a specific bacteriophage are used to solve a number of problems.

The high specificity of the lytic action of phages within the genera and families is known, therefore, a lysis test is used when there is doubt about the generic affiliation of the strain to accelerate the identification of the microbe. The study can be carried out using conventional methods for growing phages in a dense and liquid medium or a simplified method that allows one to obtain indicative data on the ability of the studied strain to be lysed by the applied phage. To perform this test, culture of the determined microbes is seeded in two test tubes with the broth; phage from 1 to 10 drops is added to one of them, depending on the lytic strength of the phage. Both tubes are placed in a thermostat for 18-20 hours, after which, according to the growth density (turbidity), the full or partial effect of the phage on the bacteria is taken into account compared to growth in the control tube.

Also, for example, phage exposure is used to determine the phagovariant of certain microbes - salmonella, typhoid, paratyphoid, staphylococcus, pseudomonas, protea - with intrapartum identification, which is very important for epidemiological practice. In epidemiological surveys, the definition of phagovars may be useful. The definition of fagovar staphylococci is carried out using phages diluted to the test dilution indicated on the label. In a Petri dish, on the surface of 1.2% agar prepared on a Hottinger digest with the addition of fresh meat water and 0.4% glucose (pH 7.6, 0.2 ml of a sterile 10% solution is added per 100 ml to the cups in the agar CaCl ₂ ) and dried for 30-40 minutes in a thermostat, pour 1-2 ml of a 4-hour broth culture, evenly distribute on the surface of the agar, allow the liquid to evaporate. Phages, one in each square, are dripped along a grid previously applied to the bottom. The bottom of the seeded cup is plotted into 22 squares according to the number of phages. It is recommended that you always follow the same phage distribution order. Leave for 3 hours at 37 ° C and then up to 18-20 hours at room temperature. Phagolysis is read in incident light on a dark background with a magnifier. Depending on the sensitivity of the culture to phage, a different degree of lysis is observed - from complete (confluent) to single sterile spots. The results are evaluated by the pluses: 4+ with confluent lysis, 3+ with confluent lysis with secondary growth, 2+ with more than 50 negative colonies. Less pronounced lysis is not considered a positive result. If this culture is not lysed with phage in test dilution, then the experiment is repeated using phages that are 100 times more concentrated. In this case, inhibitory reactions are possible that are difficult to distinguish from secondary growth, which can be observed in the case of complete lysis in the presence of phage-resistant individuals in the culture / 1 /.

Using the above-described method for evaluating phagovar, the sensitivity of the isolated microorganisms to therapeutic bacteriophages is determined.

These methods are analogues of the invention. The closest analogue (prototype) of the proposed method is a method for determining fagovar staphylococci in a dense environment.

The described methods for determining the sensitivity of microorganisms to a specific bacteriophage are long, complex and time-consuming. Test results can only be obtained after 21-24 hours.

The technical result of the invention is to increase the detection rate of the sensitivity of the test strain of the microorganism to a specific bacteriophage and to evaluate the effectiveness of the therapeutic bacteriophage by bringing the assessment conditions closer to the conditions of the macroorganism.

The result of the invention is achieved due to the fact that, under standard conditions, the monolayer of the culture of human embryo cells carries out co-cultivation for 2 hours of the test strain of the microorganism with the corresponding therapeutic bacteriophage or combined (polyvalent) bacteriophage, which includes the phage corresponding to the used test culture of the microorganism, and the result is evaluated by the degree of decrease in adhesion of the test strain of the microorganism compared with the control.

The method is as follows.

From Leighton tubes onto a coverslip with a monolayer of musculocutaneous (FCEC) or pulmonary fibroblasts (PhEC) human embryo obtained by / 2 /, the growth medium is drained and 1.8 ml of the bacteriophage preparation and 0.2 ml of the daily culture of the test strain are added at a dose of 10 ⁹ CFU / ml, obtaining a final concentration of the test strain of 10 ⁸ CFU / ml. The tubes are incubated for 2 hours at 37 ° C, then the monolayer cells are washed from non-adherent bacteria by repeated change of the Eagle medium, fixed with 96 ° ethanol, stained according to Romanovsky-Giemsa and examined microscopically, determining the degree of infection of the monolayer compared to the control. In a control tube, 1.8 ml of the support medium are co-incubated with a needle with 0.2 ml of the daily culture of the test strain at a final dose of 10 ⁸ CFU / ml. The intensity of the adhesion process is evaluated by the following indicators: 1) the adhesion index (IA) is expressed by the average number of bacterial cells in one eukaryotic cell; 2) the percentage of affected cells of the monolayer (PC%); 3) the contamination of 100 cells of the monolayer - microbial load (MN) - is determined by the formula MN = IA × PC%. The percentage of microbial adhesion is determined by the indicator of microbial load relative to the control, taken as 100%.

Example 1. Determination of the bacteriolytic activity of the preparation of pyobacteriophage (pyophage) in relation to the test strains S.aureus 209, E. coli 1257, Ps.aeruginosa 15442 in cell culture (CC) FCEC. The medium for the cultivation of test cultures of microorganisms - meat-peptone broth, for the cultivation of fibroblasts - nutrient medium Needle.

In a Leighton test tube, 1.8 ml of pyophage and 0.2 ml of daily test culture of the corresponding microorganism at a dose of 10 ⁹ CFU / ml were placed on a cover glass with a PCEC monolayer to obtain a final concentration of test strains of 10 ⁸ CFU / ml (experimental inoculum). As a control inoculum, we used a mixture of 1.8 ml of Eagle's nutrient medium with 0.2 ml of the daily test culture of the corresponding microorganism in the same dose, placed in a Layton test tube on a coverslip with a monolayer of PCEC. The tubes were incubated for 2 hours at 37 ° C, then the monolayer cells were washed from non-attached microorganisms by repeated change of the Eagle medium, fixed with 96 ° ethanol, stained with Romanovsky-Giemsa and examined under a microscope, determining the degree of infection of the monolayer and the percentage of microbe adhesion compared with the corresponding control without a pyophage in terms of microbial load (table 1).

As can be seen from the table, the percentage of adhesion of the test strains significantly decreased relative to the control, respectively: for staphylococcus - by 92.5%, for E. coli - by 96.2%, for Pseudomonas aeruginosa - by 92.1%. The decrease in the adhesive activity of the test cultures occurs due to the bacteriolytic effect of the pyophage. This is confirmed by the results of an additional test.

Inoculums were inoculated from Leighton tubes onto Petri dishes with MPA before incubation and after 2 hours of incubation at 37 ° C. The results of seeding inoculum from the corresponding control and experimental tubes are shown in table 2.

The data presented in table 2 indicate the absence of viable cells of microorganisms in both control and experimental tubes after 2 hours of incubation. The absence of microflora growth on a solid nutrient medium in the control is associated with 100% adhesion of test strains to the fibroblast cell culture in Leighton tubes. At the same time, the lack of growth of test cultures on MPA after 2 hours of incubation in the experiment indicates the death of test strains due to the bacteriolytic action of the pyophage due to the absence of adherent microorganism cells to the fibroblast cell culture in Leighton tubes.

Literature

1. Handbook of microbiological and virological research methods. // Ed. M.O. Birger. - 3rd ed., Revised. and add. - M .: Medicine, 1982. - p.118, 254.

2. K. B. Grabovskaya, A. A. Totolyan Zhurn. Microbiol. - 1977. - No. 2. - p. 32-36.

Table 1. Adhesive activity of test microorganisms in the presence of pyophage Microorganism test Test object S.aureus E.coli Ps.aeruginosa IA %PC MN % adhesion from control IA %PC MN % adhesion from control IA %PC MN % adhesion from control the control: KK + microbe 6 twenty 120 one hundred 7 thirty 210 one hundred 8 35 280 one hundred experience: KK + microbe + pyophage one 9 9 7.5 one 8 8 3.8 2 eleven 22 7.9

Table 2. Seeding with test strains of microorganisms in control and in experience Sowing on Petri dishes with MPA Microflora growth in CFU / ml S.aureus E.coli Ps.aeruginosa the control experience the control experience the control experience Prior to incubation 6 · 10 ⁸ 4 · 10 ⁸ 2 · 10 ⁸ 3 · 10 ⁸ 4 · 10 ⁸ 5 · 10 ⁸ After 2 hours of incubation at 37 ° C 0 0 0 0 0 0

Claims (1)

A method for evaluating the specific activity of bacteriophages, characterized in that under standard conditions a monolayer of the culture of human embryo cells carries out co-cultivation for 2 hours of a test strain of the microorganism with the corresponding therapeutic bacteriophage or combined (multivalent) bacteriophage, which includes the corresponding used test culture of the microorganism phage, and the result is assessed by the degree of decrease in adhesion of the test strain of the microorganism compared to the control.