Correction of vaginal dysbiosis cauced by the biofilm forming strain of Staphylococcus aureus, using probiotic based on spore microorganisms

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Oles Honchar Dnipro National University

CORRECTION OF VAGINAL DYSBIOSIS CAUSED BY THE BIOFILM FORMING STRAIN OF STAPHYLOCOCCUS AUREUS, USING PROBIOTIC BASED ON SPORE MICROORGANISMS

O.I. Makedonska, O.S. Voronkova,

Yu.S. Voronkova, A.I. Vinnikov

Annotation

Changes of microbiocenosis of different biotopes of the human body are known as dysbiosis. For the renovation of the composition of the microbiota, use the bacteriotherapeutic preparations - probiotics. The aim of the research was to study the effectiveness of the use of the mixed suspension of cells of probiotic strains Bacillus subtilis and Bacillus licheniformis for the correction of the composition of the microbiota of the vagina of white laboratory mice, induced by the intravaginal introduction of a biofilm forming strain of Staphylococcus aureus. It was shown that in cases of dysbiosis of genitourinary tract of mice, induced by the intravaginal introduction of a biofilm-forming strain of S. aureus, the use of probiotic microorganisms B. subtilis and B. licheniformis led to the tendency to normalization of the vaginal microbiota. Renovation of vaginal microbiome characterized by the increase of ratio anaerobic to anaerobic bacteria to 1: 71-1: 87 that in fact corresponded to normal index; the decrease of the frequency of determination and quantity of opportunistic bacteria, such as staphylococci and enterobacteria.

Key words: vaginal dysbiosis, microbiota, correction, probiotic, bacilli.

Анотація

О.І. Македонська, О.С. Воронкова, Ю.С. Воронкова, А.І. Вінніков

КОРЕКЦІЯ ДИСБІОЗУ ПІХВИ, ЗУМОВЛЕНОГО ВВЕДЕННЯМ ПЛІВКОУТВОЮЮЧОГО ШТАМУ ЗОЛОТИСТОГО СТАФІЛОКОКУ ІЗ ЗАСТОСУВАННЯМ ПРОБІОТИКІВ НА ОСНОВІ СПОРОВИХ МІКРООРГАНІЗМІВ

Стан порушення мікробіоти, що характеризується поняттям «дисбактеріоз», є доволі актуальною та поширеною проблемою. Для відновлення складу мікробіоти використовують бактеріотерапевтичні препарати на основі мікроорганізмів - пробіотики. Метою роботи було дослідження ефективності використання змішаної суспензії клітин пробіотичних штамів Bacillus subtilis та Bacillus licheniformis для корекції складу мікробіоти піхви білих лабораторних мишей, індукованого шляхом інтравагінального введення біоплівкотвірного штаму Staphylococcus aureus. Показано, що у випадку дисбіозу урогенітального тракту мишей, викликаного інтравагінальним введенням біоплівкотвірного штаму золотистого стафілокока, використання пробіотичних мікроорганізмів B. subtilis та B. licheniformis сприяло відновленню мікробіоти піхви. Відновлення вагінального мікробіому виражалося у зростанні індексу відношення аероби: анаероби до 1: 71-1: 87 і практично наближалося до показника норми; зниженні частоти виявлення та кількості умовно-патогенних мікроорганізмів, зокрема, стафілококів та ентеробактерій.

Ключові слова: дисбіоз піхви, мікробіота, корекція, пробіотик, бацили.

The main text

The ecological balance of the microbiocenosis of the reproductive system is an important component of human health. Thus, a disorder of the composition of the microbiocenosis of the reproductive system of a woman can lead to pregnancy loss, and can also adversely affect the child's body when passing through the birth ways of the mother [3], therefore the study of the correction of microbiota with using of preparations that are safe for humans and effective against opportunistic microorganisms. It is known that the composition of the indigenous microbiota, including the female reproductive system, formed over a long evolutionary interaction of macro- and microorganisms and today is represented by a number of such microorganisms, mainly lactobacilli, bifidobacteria and others, and temporarily include a number of transient species [3]. Among the latter, opportunistic microorganisms pose a threat, especially those capable to form biofilms, which is associated with an increase of their colonization activity [2, 15]. Among these bacteria, staphylococci are one of the most well-known. Most clinical isolates of it are able to form biofilms [2]. Their penetration into the vaginal microflora system has many significant negative consequences. In this regard, it should be noted that staphylococci quickly become resistant to antibiotics and spread these genes among cells in the microbiome, which becomes especially relevant for polycomponent microbiocenosis of the open cavities of the human body. As part of the latter, microorganisms are often prone to film formation. In a biofilm, staphylococci become significantly more resistant to antibiotics [14], and this situation requires control and treatment, which can be done with antibiotics or probiotics.

The use of bacteriotherapy drugs - probiotics, is considered to be more optimal for suppressing of opportunistic bacteria and for correcting the ecological balance of microbiota [8, 10]. Although for the normalization of the composition of the microbiota of the reproductive system the preparations based on lactobacilli are considered optimal [8], nevertheless, probiotic preparations based on spore microorganisms of the genus Bacillus, can also be considered promising.

One else prospect of use belongs to natural components for correction of microbiota. Among them can be listed plant essential oils combined with mexidol that realise the significant antimicrobial effect on opportunistic bacteria including Staphylococcus aureus [4].

It is known that bacilli realize high antagonistic activity against many bacteria and able to regulate the composition of the microbiota of various biotopes of the microorganism. The vast majority of the bacilli species are safe to macroorganism. Among different species of bacilli, some strains with high antagonistic activity were selected. This activity is more pronounced and manifests against a much wider spectrum of pathogenic and opportunistic microorganisms than in other representatives of exogenous and endogenous microbiota; they are stable during storage and environmentally safe [13].

Usually, bacilli probiotics are used for the correction of dysbacteriosis of the gastrointestinal tract, however, the high efficiency of these preparations allows one to anticipate their possible use for the correction of the microbiota of other biotopes, in particular, the vagina. Strains of bacilli - component of probiotics - B. subtilis and B. licheniformis, have a certain degree of kinship with lactic acid bacteria [7]. This provides the basis for the effective use of it in complex with other bacteria for solution of problems associated with bacterial vaginosis [11]. For the study of this issue, experimental modeling is the optimal way. Modeling of the development of dysbacteriosis provides unique opportunities: clearly measure the effect of a particular factor, study the dynamics of development, the particular composition and metabolic properties of bacteria that make up the vaginal microflora, and investigate the effects of various probiotic and other preparations on the model.

The purpose of the study was to access the effectiveness of the use of the mixed suspension of cells of probiotic strains Bacillus subtilis and Bacillus licheniformis for the correction of the composition of the microbiota of the vagina of white laboratory mice, induced by the intravaginal introduction of a biofilm forming strain of Staphylococcus aureus.

Materials and methods

For the study mice model of dysbacteriosis of the genitourinary tract was used. Studied groups of animals included female outbred white laboratory mice: control group (n=10) - animals injected with saline, experimental group 1 (n=10) - animals injected with the suspension of bacilli from “Biosporin” only, experimental group 2 (n=10) - animals, which at the first stage were injected with cells of the film-forming strain of S. aureus (strain 4v), isolated from the mouse, and in the second stage - the suspension obtained from the lyophilic preparation of the probiotic “Biosporin” (Biopharma, Ukraine) was injected.

Animals for the experiments were selected randomly from the total population of female mice aged 20-25 weeks and weighing 20-24 g, which were kept in standard conditions [1]. All research were carried out in accordance with the standards established by the Law of Ukraine No. 3447-IV “On protecting animals from ill-treatment” and the norms adopted to the “European Convention on the Protection of Vertebrate Animals used for Experimental and Scientific Purposes” (1986).

A strain S. aureus 4v used for modeling of dysbiosis of the reproductive tract was isolated from the mouse vagina. Identification was done by use of the ApiStaph test-system (bioMerieux, France). The production of pathogenicity factors were studied using standard techniques [1].

Ability to form a biofilm determined in the immunological tablet. Suspension of cells of the isolated strain was sown in the wells: 50 pl with a cell content of 10⁵ CFU/ml. The culture was incubated at 37 °C for 72 h. The formation of noticeable surface or near-bottom film growth in the well during this period was considered to be a sign of the film formation of the strain.

For modeling of a dysbacteriosis an intravaginal administration of a suspension of cells of strain S. aureus 4v isolated from the mouse was realized. The microorganisms were injected once with a dispenser into the vagina of the animals of experimental group 2 (n=10): 50 pl of cell suspension, containing 1x10⁹ CFU/ml. The control group of animals was carried out with the introduction of sterile saline (mass fraction of sodium chloride 0.9 %) - 50 pl. After the introduction of bacteria, the animals were kept in an isolated room under the same conditions as the control group. The signs of vaginal dysbacteriosis were monitored on the 10^th day after the addition of the microbial suspension according to the following features: a decrease of the aerobic/anaerobic ratio index, a decrease of the number of lactobacilli and an increase in the number of opportunistic microorganisms.

The isolation and identification of microorganisms was carried out by standard methods [1] in accordance with the signs given in the Bergey's Manual of determinative bacteriology [9].

For the study of the correction efficacy of the microbiota composition in the vagina by bacterial strains from probiotic “Biosporin”, animals with dysbacteriosis (experimental group 2 (n=10)) received intravaginally 50 ці of cell suspension from the preparation “Biosporin”, containing 1x10⁹ CFU/ml once a day for 5 days.

In parallel, healthy animals of the experimental group 1 (n=10) received only cell suspension with probiotic strains by the same scheme. The suspension was prepared in accordance with the manufacturer's recommendations. Changes in the composition of microbiocenosis were monitored for 25 days after the completion of the probiotic course. Changes in the qualitative and quantitative composition of the microbiota, including the disappearance of bacteria as components of the probiotic or, conversely, the colonization of the vagina of animals by them were evaluated.

Statistical data processing was performed by the program OriginLab 7.5 (p<0.05).

Results of the study and their discussion

A strain of staphylococcus was isolated from a randomly selected female mouse from the general group. It was identified as an S. aureus. The strain produced plasmocoagulase, lipase, lecithinase, producted hemolysins on the medium with blood (hemolysis zone diameter 5 mm). After 72 h cultivation in the plate, it produced a biofilm deposited on the walls of the three wells inoculated by cell suspension. A 50 ці suspension of daily culture of the strain, with a cell content of 1x10⁹ CFU/ml, was administered intravaginally to the animals of the experimental group 2. All animals were included in the experiments at the same time and control studying of the contents of the vagina was done in the same time periods. The composition of microbiota was studied at the qualitative (table 1) and quantitative (table 2) levels.

Table 1

The frequency of detection of bacteria of different genera from the vagina of animals of different experimental groups before and after the use of the probiotic strains of bacilli (abs/%)