RU2595423C2 - METHOD OF ESTIMATION OF ADHESIVE PROPERTIES OF CHOLERA VIBRIOS Vibrio cholerae El Tor AND Vibrio cholerae O139 ON CELL CULTURE HuTu-80 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to method of estimation of adhesive properties of cholera vibrios. Disclosed method involves following steps: a) cell monolayer HuTu-80 is prepared by growing in plastic bottles with volume of 50 ml at 100-150 thousand at 1 ml with subsequent trypsinization of culture and introduction into pen flasks with disks (disks 12.0 X 0.11 SPL Lifesciences) at 50 thousand cells in volume of 1 ml; b) preparing a bacterial suspension strains of Vibrio cholerae El Tor and Vibrio cholerae O139, which is grown on Martin agar (pH 7.7) for 18 hours, then by loop is inoculated into broth LB (pH 7.7) and maintained in a thermostat at 37 °C for three hours; c) experiment is carried out in pen flask with discs, on which formed sparse monolayer HuTu-80, added 10 mcl of bacterial suspension and 1 ml of DMEM medium with 5 % of bovine embryo serum up to concentration of cholera vibrios of 10⁶ mln. cells/ml, followed by incubation for 90 minutes at 37 °C and removal of nutrient medium, twice washed monolayer with Hanks solution with pH of 7.4, fixed with 5 % formalin for 1 hour, then dried and stained discs according to Romanovsky-Giemsa 1:5 for 40 minutes; d) by coloured discs count associated bacterial cells count is conducted, for this purpose, discs are washed with Hanks solution with pH of 7.4, removed from vials, dried, examined under microscope and adhesive properties of cholera vibrios are determined by number of vibrios, bound with 100 cells of HuTu-80, and adhesion index is calculated by formula $$AI=\frac{\sum KB}{100K},$$

where AI is adhesion index, ΣKB is total number of cells of bacteria, attached to one cell of HuTu-80 line, 100K - 100 cells line HuTu-80, results are evaluated as per obtained quantitative adhesion index, if it is more than 20, then analysed strain is highly adhesive, from 10 to 20 is mildly adhesive strain, less than 10 is low adhesive strain.

EFFECT: invention can be used in laboratory diagnostics for determination of adhesion index of cholera vibrios Vibrio cholerae El Tor and Vibrio cholerae O139 (Bengal).

1 cl, 1 tbl, 1 ex

Description

The present invention relates to medical microbiology and can be used in laboratory diagnostics to determine the adhesion index of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios (Bengal).

Currently, there are various ways to determine the level of adhesion of cholera vibrios, which is one of the main indicators of the pathogenicity of the cholera pathogen. Known methods using red blood cells (1), adhesion of cholera vibrios to the intestinal epithelium of a rabbit (2) have lost their relevance in solving this issue due to the entry into force in 2013 of the European Parliament directive on experiments with laboratory animals, according to which these methods do not meet the requirements of bioethics.

The proposed method involves the use of transplantable monolayer human cell line of adenocarcinoma of the duodenum HuTu-80, as the most effective model of human intestinal epithelial cells to determine the adhesive activity of cholera vibrios.

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, 27.08.2008), заключающийся в том, что в качестве субстрата, к которому прикрепляются холерные вибрионы, используют II группу крови человека, последнюю дефибринизируют, трижды отмывают, центрифугируют, после полученные эритроциты разводят физиологическим раствором в соотношении 1:8, ставят реакцию на стекле, после чего проводят учет реакции по среднему показателю адгезии.A known method for identifying the epidemic significance of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios by their adhesive ability (see patent RU No. 2332460, class C12Q $$\raisebox{1ex}{$\mathrm{one}$}\!\left/ \!\raisebox{-1ex}{$04$}\right.$$

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, August 27, 2008), which consists in the use of human blood group II as the substrate to which the cholera vibrios are attached, the latter is defibrinized, washed three times, centrifuged, after the obtained red blood cells are diluted with saline in a ratio of 1: 8, the reaction is glass, after which the reaction is recorded according to the average adhesion index.

However, the disadvantage of this method in the laboratory is the instability of red blood cells of the II group of human blood, the difficulty in obtaining and standardizing them.

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, C12N¹/₁₂₀, 20.12.2013), включающий подготовку бактериальной суспензии, отделение полученной биомассы бактерий центрифугированием, разведение полученной биомассы в физиологическом растворе, подготовку монослоя клеток СаСо-2, внесение бактериальной культуры, культивирование клеток, промывку физиологическим раствором, снятие монослоя с бактериальными клетками и подсчитыванием количества связанных с 1000 клетками СаСо-2 бактериальных клеток, при этом бактерии относятся к высокоадгезивным, если количество связавшихся клеток составляет от 1010 до 3000, среднеадгезивным от 210 до 1000, к низкоадгезивным от 0 до 200.For the prototype, a method was selected for determining the adhesive properties of Enterococcus bacteria using the CaCo-2 cell line (see patent RU No. 2501861, class C12Q $$\raisebox{1ex}{$\mathrm{one}$}\!\left/ \!\raisebox{-1ex}{$02$}\right.$$

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, C12N _^1/120, 20.12.2013), comprising preparing a bacterial suspension, separating the resulting bacterial biomass by centrifugation, the biomass obtained dilution in saline preparation monolayer CaCo-2 cells, introduction of the bacterial culture, culturing the cells, washing with saline solution, removing the monolayer bacterial cells and counting the number of bacterial cells bound to 1000 CaCo-2 cells, while the bacteria are highly adhesive if the number of cells bound is from 1010 to 3 000, medium adhesive from 210 to 1000, to low adhesive from 0 to 200.

The disadvantage of the prototype is that the known method cannot be used for monitoring cholera, since the preparatory phase of CaCO-2 for the experiment is long (the culture grows within 14 days) and time-consuming.

In this case, the CaCO-2 culture during cultivation requires the introduction of additional nutrients into the growth medium, which leads to a rise in the cost of research.

In addition, the use of trypsin and versene at the stage of removing the monolayer with attached bacterial cells adversely affects the cell wall of cholera vibrios, which leads to large errors in research, and the serial dilution method affects the accuracy of the results.

The technical task of the invention is to develop a new method having high accuracy in detecting the adhesive ability of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios using HuTu-80 cell culture.

This object is achieved in that the method for evaluating the adhesive properties of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios in HuTu-80 cell culture comprises the following steps:

a) prepare a monolayer of HuTu-80 cells by growing them in plastic bottles of 50 ml, 100-150 thousand per 1 ml followed by trypsinization of the culture and adding it to penflon with disks (disks 12.0 X 0.11 SPL Lifesciences ) 50 thousand cells in a volume of 1 ml;

b) prepare a bacterial suspension of the Vibrio cholerae El Tor and Vibrio cholerae O139 strains, which are grown on Marten agar (pH 7.7) for 18 hours, after which the loop is seeded in LB broth (pH 7.7) and incubated at 37 ° C for three hours;

c) the experiment is carried out in pen-bottles with disks on which a diluted HuTu-80 monolayer was formed, 10 μl of bacterial suspension and 1 ml of DMEM medium with 5% bovine fetal serum are added to a concentration of cholera vibrios of 106 m.k. / ml, then incubation is carried out for 90 minutes at 37 ° C, the nutrient medium is removed, washed twice with a monolayer of Hanks solution pH 7.4, fixed with 5% formalin for 1 hour, then dried and stained with Romanovsky-Giemsa disks 1: 5 in for 40 minutes;

d) the stained bacterial cells are counted on stained discs, for this, the discs are washed with Hanks pH 7.4, removed from the vials, dried, examined under a microscope and the adhesive properties of cholera vibrios are determined by the number of vibrios bound to 100 HuTu-80 cells, and calculate the adhesion index by the formula

где $$\mathrm{AND}\mathrm{BUT}=\frac{\sum \mathrm{TO}B}{\mathrm{one\; hundred}\mathrm{TO}},$$

Where

IA - adhesion index,

∑KB - the total number of bacterial cells attached to one cell of the HuTu-80 line,

100K - 100 cells of the HuTu-80 line,

evaluate the results according to the obtained quantitative index of adhesion, if it is more than 20, conclude that the studied strain is highly adhesive, from 10 to 20 is medium adhesive, less than 10 is a low adhesive strain.

The method is as follows.

To carry out the method, a HuTu-80 cell line (human duodenum) is required, which is obtained from the Russian collection of vertebrate cell cultures of the Institute of Cytology RAS (St. Petersburg).

The proposed method includes the following steps:

1) preparatory;

a) thawing the cell line and its adaptation to the conditions of the experiment;

b) cultivation of strains of Vibrio cholerae El Tor and Vibrio cholerae O139 in nutrient media for experiments;

2) conducting an experiment - the interaction of cholera vibrio cells with a transplanted monolayer human duodenal adenocarcinoma cell line HuTu-80;

3) taking into account the results of exposure to Vibrio cholerae cells with the transplanted monolayer human duodenal adenocarcinoma cell line HuTu-80.

Stage 1, a - Ampoules with cell culture are removed from the bio-storage and thawed at a temperature of 37 ° C, keeping the ampoule in a water bath for 2-3 minutes. Using the test of intravital staining with trypan blue, their viability is determined, the latter should be at least 80-85%.

The suspension of monolayer cells was transferred to a test tube with 199 medium for washing away from the impurities of the protective medium and centrifuged at 1000 rpm for 10 minutes, the supernatant was removed, the cell pellet was resuspended in a complete growth medium at pH 7.5.

Cells are seeded in plastic bottles with a volume of 50 ml, 100-150 thousand per 1 ml. Cultivation is carried out in a CO ₂ incubator (Naure) at a temperature of 37 ° C and a CO ₂ concentration of 5%, with a seeding frequency of 2-3 times a week. Trypsin and versene solutions in a 1: 3 ratio are used to remove the HuTu-80 cell monolayer. In the process of replicating a cell culture using an inverted microscope (Nikon), their morphology and the absence of extraneous bacterial microflora are evaluated.

For the experiment, a fresh trypsinized cell culture of the HuTu-80 line is used. 50 thousand cells in a volume of 1 ml are introduced into pen-bottles with disks (12.0 X 0.11 SPL Lifesciences disks). The next day, using an inverted microscope, the formation of a rarefied monolayer is evaluated.

Stage 1, b - Vibrio cholerae cultures are prepared immediately before the experiment. For this, the strains are grown on Marten agar (pH 7.7) for 18 hours, after which time one standard loop (d = 2 mm) of the test culture is inoculated into LB broth (Luria-Bertani) (pH 7.7), maintained in the thermostat at a temperature of 37 ° C for three hours. After that, with the help of densimeter (Erba Lachema) determine the concentration of cholera vibrios in broth cultures. All cultures were adjusted to a concentration of 10 ⁸ m.k. / ml.

The second stage is to determine the adhesive properties of cholera vibrios, for this, 10 μl of standardized suspension are added to 1 ml of DMEM medium with 5% bovine serum to obtain a final concentration of 10 ⁶ MK / ml of the tested cholera vibrio strains and added to penflon bottles with disks (12.0 X 0.11 SPL Lifesciences disks) on which a sparse HuTu-80 monolayer is formed. Incubated for 90 minutes at 37 ° C in a CO ₂ incubator (Naure), then the nutrient medium was removed and the diluted monolayer was washed twice with Hanks solution (pH 7.4) from cholera vibrios that did not contact the monolayer and fixed with 5% formalin for 1 hour. compliance with biological safety requirements, dry and stain discs according to Romanovsky - Giemsa 1: 5 for 35-40 minutes. The colored discs are washed three times with Hanks' solution (pH 7.4) and removed from the vials.

The third step, recording results, includes viewing discs under a Nikon light microscope (magnification 100X10). Browse discs always begin with control. The adhesive properties of bacteria are determined by the number of vibrios that bind to 100 HuTu-80 cells and the adhesion index is calculated by the formula

где $$\mathrm{AND}\mathrm{BUT}=\frac{\sum \mathrm{TO}B}{\mathrm{one\; hundred}\mathrm{TO}},$$

Where

IA - adhesion index,

∑KB - the total number of bacterial cells attached to one cell of the HuTu-80 line,

100K - 100 cells of the HuTu-80 line.

Assessment of the adhesion index: more than 20 is a highly adhesive strain, from 10 to 20 is a medium adhesive strain, less than 10 is a low adhesive strain.

The use of the method for assessing the adhesive properties of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios was confirmed at the Rostov Anti-Plague Institute (Rostov-on-Don) on 20 strains.

Example

Adhesive properties were determined for Vibrio cholerae El Tor 18775, Vibrio cholerae El Tor 18777, Vibrio cholerae O139 17258 strains of cholera vibrios isolated from humans and from environmental objects and characterized by the presence or absence of cholera toxin genes and toxin-regulated adhesion peels.

The experiment is conducted in the biological safety box (BMB) of class II. Vibrio cholera cultures are prepared immediately before the experiment. For this, the strains are grown on Marten agar (pH 7.7) for 18 hours, after this period one standard loop (d = 2 mm) of the test culture is inoculated into LB broth (pH 7.7), incubated at 37 ° C for 3 hours. After that, with the help of densimeter (Erba Lachema) determine the concentration of cholera vibrios in broth cultures. All cultures are standardized to a concentration of 10 ⁸ microns. cells / ml In 1 ml of DMEM medium with 5% fetal bovine serum, 10 μl of standardized suspension was added to obtain a final concentration of vibrios of 10 ⁶ mc / ml. They are introduced into pen-bottles with disks (12.0 X 0.11 SPL Lifesciences disks) in which a diluted HuTu-80 monolayer is present.

An intact culture penflacon serves as a negative control. All samples are incubated in a CO ₂ incubator at 37 ° C for 90 minutes. After this time, the experimental and control samples are washed with Hanks solution pH 7.4 from non-adherent cells of cholera vibrios and fixed with 5% formalin for 1 hour to comply with biological safety requirements, the disks are dried and stained according to Romanovsky - Giemsa 1: 5 for 35-40 minutes . The colored discs are washed three times with Hanks solution pH 7.4, removed from the vials, dried, viewed under a light microscope.

The adhesive properties of cholera vibrios are determined by the number of vibrios that bind to 100 HuTu-80 cells, and the adhesion index is calculated by the formula.

The tested strains of cholera vibrios differed in the adhesion index, which is presented in the calculations and the table.

Strain Vibrio cholerae El Tor 18775 ∑ KB = 2036.

- высокоадгезивный $$\mathrm{AND}\mathrm{BUT}=\frac{\sum \mathrm{TO}B}{\mathrm{one\; hundred}\mathrm{TO}}=2036/\mathrm{one\; hundred}=20.36$$

- highly adhesive

Strain Vibrio cholerae El Tor 18777 ∑ KB = 1017.

- среднеадгезивный $$\mathrm{AND}\mathrm{BUT}=\frac{\sum \mathrm{TO}B}{\mathrm{one\; hundred}\mathrm{TO}}=1017/\mathrm{one\; hundred}=10.17$$

- medium adhesive

Strain Vibrio cholerae O139 17258 ∑ KB = 511.

- низкоадгезивный $$\mathrm{AND}\mathrm{BUT}=\frac{\sum \mathrm{TO}B}{\mathrm{one\; hundred}\mathrm{TO}}=211/\mathrm{one\; hundred}=5.11$$

- low adhesive

Thus, the method for evaluating the adhesive properties of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios is based on the interaction of bacteria with receptors located on the cell wall of human duodenal adenocarcinoma HuTu-80 epithelial cells.

The advantages of the proposed method are that the HuTu-80 cell culture used is standard, homogeneous and has a fixed phenotype, experiments with the culture can be repeated many times, in addition, intravital observation of the adhesion process using a microscope is possible, which excludes experiments on animals. The new method allows you to study the investigated properties directly on human epithelial cells, which gives the basis for extrapolation of the results to the body as a whole. This method uses human cells, not animal cells, which makes it possible to determine the adhesiveness of bacteria with greater accuracy.

Information sources

1. Sappo S.G., Urbanovich L.Ya., Kolesnik B.C. "The use of red blood cells to detect the adhesive ability of vibrios." The collection "Modern aspects of the prevention of zoonotic infections", Ministry of Health of the USSR, Irkutsk, 1984, pp. 126 -127.

2. Guidelines for the quantitative assessment of the adhesive activity of cholera vibrios in vitro (Rostov-on-Don, 1996).

Claims (1)

A method for evaluating the adhesive properties of Vibrio cholerae El Tor and Vibrio cholerae O139 cholera vibrios on a HuTu-80 cell culture, comprising the following steps:
a) prepare a monolayer of HuTu-80 cells by growing them in plastic bottles of 50 ml, 100-150 thousand per 1 ml followed by trypsinization of the culture and adding it to penflon with disks (disks 12.0 X 0.11 SPL Lifesciences ) 50 thousand cells in a volume of 1 ml;
b) prepare a bacterial suspension of strains of Vibrio cholerae El Tor and Vibrio cholerae O139, which are grown on Marten agar (pH 7.7) for 18 hours, after which the loop is seeded in LB broth (pH 7.7) and incubated in a thermostat at 37 ° C for three hours;
c) the experiment is carried out in pen-bottles with disks on which a diluted HuTu-80 monolayer was formed, 10 μl of bacterial suspension and 1 ml of DMEM medium with 5% bovine fetal serum are added thereto, to a concentration of cholera vibrios of 10 ⁶ m.k. / ml, then carry out incubation for 90 minutes at 37 ° C, remove the nutrient medium, wash the monolayer with Hanks solution pH 7.4 twice, fix with 5% formalin for 1 hour, then dry and stain the discs according to Romanovsky-Giemsa 1: 5 for 40 minutes;
d) the stained bacterial cells are counted on stained discs, for this, the discs are washed with Hanks pH 7.4, removed from the vials, dried, examined under a microscope and the adhesive properties of cholera vibrios are determined by the number of vibrios bound to 100 HuTu-80 cells, and calculate the adhesion index by the formula:

Where
IA - adhesion index,
ΣKB - the total number of bacterial cells attached to one cell of the HuTu-80 line,
100K - 100 cells of the HuTu-80 line,
evaluate the results according to the obtained quantitative index of adhesion, if it is more than 20, conclude that the studied strain is highly adhesive, from 10 to 20 is medium-adhesive strain, less than 10 is a low-adhesive strain.