RU2500411C2 - Method for individual selection of preparations containing probiotic lactic bacterial strains for effective intravaginal therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely gynaecology and may be used for individual selection of the preparations containing the probiotic lactic bacterial strains for effective intravaginal therapy. For this purpose, vaginal epithelial cells are recovered from the patient, released from the accompanying microflora. That is followed by preparing an epithelial cell suspension in a culture medium, and mixed with a suspension of thermally-activated probiotic lactic bacterial strains. Then, the suspension is incubated; the epithelial cell culture fluid filtrate is prepared and added to the suspension of the tested probiotic lactic bacterial strains in ratio 1:7. Concurrently, a reference of the mixture of the epithelial cell culture medium and the suspension of the tested probiotic lactic bacterial strains in ratio 1:7 is prepared. The test and reference samples are incubated, measured for optical density; and a degree of biomass increase in the test sample is related to that in the reference. The preparation containing the probiotic lactic bacterial strains the biomass increase of which under the influence of patient's vaginal epithelial cells is stimulated most is selected form the effective intravaginal therapy.

EFFECT: invention enables the individual selection of the preparations containing the probiotic lactic bacterial strains for the effective intravaginal therapy.

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Description

The invention relates to microbiology and medicine and can be used in the research work of the research institute, the practical work of bacteriological laboratories in clinical institutions and obstetric and gynecological practice for the individual selection of preparations containing probiotic strains of lactobacilli for intravaginal therapy.

Probiotic strains are bacterial pathogens for humans that have antagonistic activity against pathogenic and opportunistic bacteria and restore normal microflora [Food and Agriculture Organization of the United Nations and World Health Organization. 2001, posting date. Regulatory and clinical aspects of dairy probiotics. Food and Agriculture Organization of the United Nations and World Health Organization Expert Consultation Report. Food and Agriculture Organization of the United Nations and World Health Organization Working Group Report. (Online.)].

The main goal of probiotic therapy is to restore normal microflora. To restore the microflora of the female reproductive tract, intravaginal probiotic therapy is used [Reid, G., K. Millsap, and A.W. Bruce. 1994. Implantation of Lac-tobacillus casei var rhamnosus into vagina. Lancet 344: 1229.]. The effectiveness of intravaginal probiotic therapy is judged by an increase in vaginal colonization with lactobacilli.

One of the ways to increase the effectiveness of probiotic therapy is to search for probiotic strains with pronounced adhesive and antagonistic properties.

It is known that the condition for successful probiotic therapy is the correspondence of the source of the allocation of the probiotic strain to the biotope in which its use is planned. The nature of the microflora of each individual has its own characteristics, due to the polymorphism of the genes of the macroorganism, which determine the expression of pattern-recognizing receptors, bactericidal substances and. etc. [Linhares I.M., Giraldo P.C., Baracat E.C. New findings about vaginal bacterial flora. Rev. Assoc. Med. Bras. 2010, 56 (3): 370-4.]. This determines the need for individualization of probiotic therapy.

The literature describes options for implementing this approach. A known method of selecting a probiotic preparation in which the prescribed probiotic preparation is proposed to be tested by a method similar to the determination of antibiotic sensitivity for antagonism in relation to the pathogen and normoflora of an individual isolated from a “problem” biotope [Gonchar N.V., Berezina L.V., Tikhomirova O.V. . The choice of a probiotic for the rational treatment of Klebsiella infection in children // Zh. microbiol. - 2009. - No. 2. - S.85-89 .; RF patent No. 2428468 IPC C12N 1/20, А61К 35/74, 09/10/2011, Bull. No. 25.]. The selection criterion is the nature of the interaction in which the probiotic preparation suppresses pathogenic microorganisms, and there is no negative effect on the development of normal microflora.

There are methods for the individual selection of preparations containing probiotic strains, in which, in addition to the foregoing, take into account the modification of the antagonism and / or growth properties of the individual normal flora under the influence of the probiotic strain [AV Semenov Characteristic of the relationship between probiotic and autochthonous microorganisms and the algorithm for the individual selection of probiotics // Kazan medical journal. - 2011. - T.2. - No. 6. - S.792-795].

However, the above methods are based on determining the interaction of probiotic strains with the microflora of the human body and do not allow for the interaction of the probiotic strain with macroorganism cells, which largely determines the formation of microflora. These are primarily mucosal epithelial cells, which, due to their position on the border of internal and external environments, contacts with both normoflora and allochthonous microorganisms, as well as their ability to regulate mucosal immunity, play a key role in the functioning of mucous microbiocenoses [Wira C.R ., Grant-Tschudy KS, Crane-Godreau MA Epithelial Cells in the Female Reproductive Tract: a Central Role as Sentinels of Immune Protection. American Journal of Reproductive Immunology. 2005, 53 (2): 65-76.].

A known method of individual selection of preparations containing probiotic strains of lactobacilli, based on a comparative assessment of the degree of adhesion of probiotic strains of lactobacilli to vaginal epithelial cells of the patient. [A.G. Boytsov, S.V. Rishchuk, Yu.Yu. Ilyasov, T.A. Grechaninova Adhesion of lactobacilli to the cells of the vaginal and buccal epithelium // Bulletin of the St. Petersburg Medical Academy. I.I. Mechnikov. - 2004. - No. 4 (5) - S.191-193.].

However, this method allows you to evaluate only the property necessary for the initial stage of colonization, and a priori expressed in all probiotic strains. The implementation of adhesive activity does not depend on the viability, metabolic, reproductive activity and antagonistic potential of bacterial cells, which determine the success of the colonization of the biotope [Mayansky, AN Pathogenetic microbiology: a guide / A.N. Mayansky. - N. Novgorod: NGMA, 2006 .-- 520 p. - c7].

The relevance of developing a method for the individual selection of drugs containing probiotic strains of lactobacilli for intravaginal therapy is due to the need to increase the effectiveness of probiotic therapy, which can be used to correct dysbiotic and inflammatory conditions of the reproductive tract of women and will help preserve the reproductive health of women.

The technical result to which the invention is directed is to create a method that allows, on the basis of determining the interaction of probiotic strains of lactobacilli with vaginal epithelial cells, to individually select preparations containing probiotic strains of lactobacilli for effective intravaginal therapy.

To achieve the indicated technical result, in the claimed method of individual selection of preparations containing probiotic strains of lactobacilli for effective intravaginal therapy, vaginal epithelial cells are isolated from the patient, they are released from concomitant microflora, a suspension of epithelial cells is prepared in the culture medium and mixed with a suspension of washed thermally inactivated probiotic strains, lactobacilli strains are incubated; get the filtrate of the culture fluid of epithelial cells and add it in a ratio of 1: 7 to suspensions of the tested probiotic strains of lactobacilli, in parallel prepare a control from a mixture of the culture medium of epithelial cells and suspensions of the tested probiotic strains in a ratio of 1: 7, the experimental and control samples are incubated, the absorbance is measured, the degree of biomass increase in the experimental sample is calculated relative to the control and for effective intravine therapies choose a drug containing probiotic strains of lactobacilli, whose biomass increase, under the influence of the patient's vaginal epithelial cells, with imuliruetsya most pronounced.

The technical result from the implementation of the invention is expressed in the creation of a method for the individual selection of probiotic preparations containing lactobacilli for effective intravaginal therapy, based on the identification of probiotic strains whose biomass increase under the influence of vaginal epithelial cells of the patient is most pronounced.

Earlier, we obtained data on the ability of vaginal epithelial cells and their secretory products to differentially affect the growth properties and antagonistic activity of various representatives of the vaginal microbiocenosis and a method was developed for determining this effect [gender. decision on 3.2010144750 / 10 of 01.16.2012, publ. applications 02.10.2011 Bull. No. 4].

The authors experimentally established the information content of determining the effect of the patient's previously stimulated thermally inactivated probiotic strains of lactobacilli vaginal epithelial cells on the growth of these probiotic strains with the aim of individually selecting probiotic preparations containing lactobacilli for effective intravaginal therapy.

The authors conducted a study aimed at identifying the relationship between the effectiveness of intravaginal probiotic therapy and the peculiarities of the interaction of the patient's vaginal epithelial cells with a probiotic strain of lactobacilli. The study used a probiotic strain of Lactobacillus acidophilus of vaginal origin, which is part of the drug for intravaginal use of Ecofemin (Pharma-vinci, Denmark).

To conduct studies in 24 patients suffering from vaginal dysbiosis, vaginal epithelial cells were obtained from the lateral vaginal fornix, they were released from concomitant microflora, incubated for one hour in Hanks medium, with the addition of the cefuroxime and lysozyme antibiotic, washed three times with a ten-fold volume of physiological saline, and then prepared suspension with a concentration of 10 ⁶ cells / ml.

To stimulate epithelial cells, a probiotic strain of Lactobacillus acidophilus, inactivated by heating to 75 ° C, was added to this suspension over 20 minutes, observing the ratio of epithelial cell: microorganism of 1:10. To assess the effect of epithelial cells on the growth and antagonism of the probiotic strain, the filtrate of a 4 hour culture fluid of stimulated epithelial cells was added in a 1: 7 ratio to the suspension of the tested probiotic strain of lactobacilli. The change in the growth properties of the probiotic strain was determined photometrically (at λ = 540 nm) and the cup method by counting the number of colony forming units (CFU) after plating on MRS agar.

The antagonistic activity of the probiotic strain Lactobacillus acidophilus was studied using the bactericidal test method of the culture fluid of probiotic strains [Semenov A.V., Sgibnev A.V., Cherkasov S.V., Bukharin O.V. Microbial regulation of antagonistic activity of bacteria. Bull. exp biol. honey. 2007; 11: 545-48.], Obtained under the influence of extracellular secretory products of vaginal epithelial cells. The control was the culture fluid of probiotic strains obtained under the influence of the culture medium of epithelial cells.

Changes in the biomass growth rate and antagonistic activity of the probiotic strain Lactobacillus acidophilus under the influence of vaginal epithelial cells, previously stimulated with thermally inactivated probiotic strains were unidirectional and correlated with each other (r = 0.81, p <0.05), therefore, we use both of these properties for the selection of the drug for intravaginal probiotic therapy considered inappropriate and further studies were limited to studying the effect of epithelial cells on the growth properties of the probiotic strain.

According to the results of determining the effect of vaginal epithelial cells on the growth properties of the probiotic strain Lactobacillus acidophilus, all patients participating in the study with vaginal dysbiosis were divided into three groups:

1 - vaginal epithelial cells stimulate the growth properties of the probiotic strain of Lactobacillus acidophilus;

2 - vaginal epithelial cells have an indifferent effect, i.e. change the biomass increase of the probiotic strain of Lactobacillus acidophilus by no more than 5%;

3 - vaginal epithelial cells inhibit the growth of biomass of the probiotic strain Lactobacillus acidophilus.

The first group included 14 women, which amounted to 58.3% of the total number of patients, the second - 6 patients (25.0%), and the third - 4 patients (16.7%).

After determining the effect of vaginal epithelial cells on the growth properties of the probiotic strain Lactobacillus acidophilus, probiotic therapy with Ecofemin was performed according to the manufacturer's recommendations (Pharma-vinci, Denmark) by intravaginal administration of the capsule 2 times a day for 6 days.

A comparative evaluation of the effectiveness of probiotic therapy in the studied groups was carried out 7 days after the completion of the course of therapy, and since the microflora of the genital tract of a woman is influenced by cyclically changing parameters of existence, and the ability to adapt to these constant changes is a prerequisite for maintaining normal vaginal flora, we evaluated vaginal contamination with lactoflora a second time - in the first phase of the menstrual cycle following intravaginal probiotic therapy but.

The effectiveness of probiotic therapy was determined by restoring the contamination of the vaginal mucosa by lactobacilli (PMO≥10 ⁶ ). The obtained data were processed statistically: a comparison of the average values of the indicators was carried out according to the paired student criterion in the case of dependent samples and the student t-test for independent samples. The differences were considered significant if the probability of error p was less than 0.05 [Lakin R.F. Biometrics. M., High School, 1990.].

The research results are presented in diagrams (figure 1 and figure 2).

Figure 1 shows the contamination of the vagina with lactobacilli on the 7th day after the completion of probiotic therapy. Vertically indicated groups of patients, horizontally, the frequency of isolation of lactobacilli (%). The black column indicates PMO of lactobacilli ≤10 ³ ; dark gray columns - PMO of lactobacilli 10 ⁴ ; light gray columns - PMO of lactobacilli 10 ⁵ ; white columns - PMO of lactobacilli ≥10 ⁶ .

From the presented diagram (Fig. 1), it is seen that a week after the end of treatment, in patients whose vaginal epithelial cells stimulated an increase in the biomass of the probiotic strain Lactobacillus acidophilus (group 1), in 85.7% of cases there was a restoration of vaginal contamination with lactoflora, in the rest (14.3%), an increase in the indicator of microbial contamination of the vagina by lactobacilli was also observed, but not exceeding 10 ⁵ CFU / ml.

In the 2nd group of patients whose vaginal epithelial cells influenced the growth of the probiotic strain Lactobacillus acidophilus indifferently, lactoflora recovery was observed only in 16.7% of cases, and in patients of the 3rd group, whose vaginal epithelial cells inhibit the growth of the biomass of the probiotic strain Lactobacillus acidophilus lactobacillus, there was no recovery.

Figure 2 shows the contamination of the vagina with lactobacilli in the first phase following the probiotic treatment of the menstrual cycle. Vertically indicated groups of patients, horizontally, the frequency of isolation of lactobacilli (%). The black column indicates PMO of lactobacilli ≤10 ³ ; dark gray columns - PMO of lactobacilli 10 ⁴ ; light gray columns - PMO of lactobacilli 10 ⁵ ; white columns - PMO of lactobacilli ≥10 ⁶ .

As can be seen from the diagram (Fig. 2), after one menstrual cycle, the situation worsened somewhat while maintaining the previous trend: the normal number of lactobacilli was recorded only in the 1st group of patients whose vaginal epithelial cells stimulated an increase in the biomass of the probiotic strain Lactobacillus acidophilus, although, the proportion of such patients decreased from 85.7% to 64.3%. This was due to an increase in the number of women with moderate deficiency of lactoflora. A pronounced deficiency of normoflora was not recorded in any woman in this group. On the contrary, in patients of the third group during one menstrual cycle the effect of intravaginal probiotic therapy was completely leveled, and the number of lactobacilli returned to the previous level.

Thus, the data obtained allow us to evaluate the interaction of probiotic strains of lactobacilli with vaginal epithelial cells as the basis for the formation of microbiocenoses, which can be used for the individual selection of preparations containing probiotic strains of lactobacilli for effective intravaginal probiotic therapy.

In order to confirm the possibility of using the proposed method, we studied the effect of vaginal epithelial cells obtained from specific patients on the growth properties of different probiotic strains of lactobacilli isolated from Lactobacterin (Perm), Ginoflor (Medinowa, Germany), and Ecofemin ( Pharma-vinci, Denmark), “Acylact” (Ferment LLC, Russia), “Lactonorm” (LECCO CJSC, _Russia). The research results are presented in the table.

The results indicate the individual nature of the interaction of the vaginal epithelial cells of a particular patient with different probiotic strains of lactobacilli. From a practical point of view, this confirms the need for individual selection of preparations containing probiotic strains of lactobacilli for effective intravaginal probiotic therapy.

The method is as follows.

1. From the side walls of the vagina of a particular patient for which selection is being performed, vaginal epithelial cells are taken with a Volkman spoon.

2. Obtaining vaginal epithelial cells exempt from concomitant microflora, incubating for one hour in Hanks medium, with the addition of 50 μg / ml antibiotic and 10 μg / ml lysozyme, and washed three times with ten times the volume of saline.

3. Prepare a suspension of epithelial cells in Hanks at a concentration of 10 ⁶ cells / ml.

4. The daily culture of the tested probiotic strains of lactobacilli is inactivated by heating to 75 ° C for 20 minutes, washed and a suspension is prepared with a concentration of 10 ⁸ cells / ml in physiological saline.

5. To 1 ml. Suspensions of epithelial cells add 0.1 ml. a suspension of thermally inactivated probiotic strains of lactobacilli to create a ratio of epithelial cell: microorganism - 1:10, incubated at 37 ° C for 4 hours and collect the culture fluid.

6. The resulting culture fluid of stimulated epithelial cells is filtered and added in a ratio of 1: 7 to a suspension of the tested probiotic strains of lactobacilli in MRS medium with a concentration of 10 ⁶ cells / ml.

7. In parallel, a control is prepared from the medium for the cultivation of epithelial cells and suspension of the tested probiotic strains of lactobacilli in a ratio of 1: 7.

8. The experimental and control samples are incubated for 4-6 hours at 37 ° C, stirring occasionally.

9. Measure the optical density in the experimental and control samples, calculate the degree of biomass increase in the probiotic strain of lactobacilli in the experimental sample relative to the control.

10. A drug containing a probiotic strain whose biomass increase, under the influence of vaginal epithelial cells, is stimulated by the patient is most strongly recommended for effective intravaginal probiotic therapy.

Examples of specific performance of the method.

Example 1

We conducted an individual selection of a drug containing probiotic strains of lactobacilli for intravaginal therapy for patient O.

From the lateral wall of the patient’s vagina, O. Volkman’s spoon received vaginal epithelial cells. To free them from concomitant microflora, epithelial cells were incubated for one hour in Hanks medium, with the addition of 50 μg / ml cefuroxime and 10 μg / ml lysozyme, then washed three times with ten times the volume of saline and a suspension of epithelial cells in Hanks medium was prepared at a concentration of 10 ⁶ cells / ml., Wash and prepare a suspension with a concentration of 10 ⁸ cells / ml in physiological saline.

The daily cultures of probiotic strains of lactobacilli isolated from Lactobacterin (Perm), Ginoflor (Medinowa, Germany), and Ecofemin (Pharma-vinci, Denmark) were thermally inactivated by heating for 20 minutes and they were prepared with suspension from concentration of 10 ⁸ cells / ml in physiological saline.

To 1 ml. Suspension of epithelial cells was added 0.1 ml. suspensions of thermally inactivated probiotic strains of lactobacilli. Incubated for 4 hours at 37 ° C.

Then the culture fluid of the epithelial cells was collected, filtered through bacterial filters and added in a ratio of 1: 7 to a suspension of the tested probiotic strains of lactobacilli in MRS medium with a concentration of 10 ⁶ cells / ml. The control was prepared from a mixture of medium for the cultivation of epithelial cells and suspension of the tested probiotic strains of lactobacilli in a ratio of 1: 7.

During 4-hour incubation at 37 ° C, the optical density (at λ = 540 nm) was measured in experimental and control samples, and the degree of biomass increase in the experiment relative to the control was calculated.

The increase in the biomass of lactobacilli isolated from the Lactobacterin preparation in relation to the control was 83%, lactobacilli isolated from the Ginoflor drug was 110%, and lactobacilli isolated from the Ecofemin preparation was 143%. Consequently, the vaginal epithelial cells of patient O. have the most pronounced stimulating effect on the biomass growth of the probiotic strain of lactobacilli contained in the Ecofemin preparation, and it is precisely this that must be chosen for effective intravaginal probiotic therapy.

Example 2

We conducted an individual selection of the drug for intravaginal probiotic therapy for patient C.

According to the claimed method, analogously to example 1, the effect of vaginal epithelial cells, previously stimulated with thermally inactivated probiotic strains of lactobacilli on the growth of biomass of these tested probiotic strains was determined.

The biomass of the probiotic strain of lactobacilli isolated from the Lactobacterin preparation was 89% relative to the control, the probiotic strain of lactobacilli isolated from the Ginoflor drug was 133%, and the probiotic strain of lactobacillus isolated from Ecofemin was 103 %

Therefore, the vaginal epithelial cells of patient C. have the most pronounced stimulating effect on the growth of the probiotic strain of lactobacilli contained in the Ginoflor preparation, and it is precisely this that must be chosen for effective intravaginal probiotic therapy.

Example 3

An individual selection of the drug for intravaginal probiotic therapy for patient X was carried out. According to the claimed method, similarly to example 1, the effect of vaginal epithelial cells, previously stimulated with heat-inactivated probiotic strains of lactobacilli on the biomass increase of the tested probiotic strains of lactobacilli was determined.

The biomass of the probiotic strain of lactobacilli isolated from the Lactobacterin preparation was 92% relative to the control, the probiotic strain of lactobacilli isolated from the Ginoflor drug was 83%, and the probiotic strain of lactobacillus isolated from the Ecofemin preparation was 96%. Therefore, the vaginal epithelial cells of patient X. do not have a stimulating effect on the growth of the tested probiotic strains of lactobacilli. For effective intravaginal probiotic therapy in patient X. these probiotic strains cannot be recommended.

Thus, the claimed method allows, on the basis of determining the interaction of probiotic strains of lactobacilli with vaginal epithelial cells, to individually select preparations containing probiotic strains of lactobacilli for effective intravaginal therapy.

Table The effect of vaginal epithelial cells on the biomass increase of probiotic strains of lactobacilli isolated from different drugs (in% relative to control) Samples of vaginal epithelial cells The biomass increase of probiotic strains of lactobacilli isolated from drugs Lactobacterin (%) Gynoflor (%) Ecofemin (%) Acylact (%) Lactonorm (%) one 87 134 108 105 106 2 92 102 118 101 135 3 101 96 143 86 99 four 75 128 102 99 112 5 96 105 115 115 89 6 89 97 129 111 87 7 99 105 116 93 127 8 one hundred 85 109 141 69 9 105 98 111 110 135 10 76 140 106 112 101 eleven 118 96 110 102 106 12 91 93 87 97 89

Claims (1)

The method of individual selection of preparations containing probiotic strains of lactobacilli for effective intravaginal therapy, which consists in isolating vaginal epithelial cells from the patient, releasing them from the concomitant microflora, preparing a suspension of epithelial cells in a culture medium and mixing them with a suspension of washed thermally inactivated probiotic strains, incubating lactobacilli, get the filtrate of the culture fluid of epithelial cells and add it in a ratio of 1: 7 to the suspension of the tested probiotic staff mm of lactobacilli, in parallel, a control is prepared from a mixture of the culture medium of epithelial cells and suspensions of the tested probiotic strains in a ratio of 1: 7, the experimental and control samples are incubated, the optical density is measured, the degree of biomass increase in the experimental sample is calculated in relation to the control one, and the drug is chosen for effective intravaginal therapy containing probiotic strains of lactobacilli, whose biomass increase under the influence of the patient's vaginal epithelial cells is most pronouncedly stimulated.

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