OF LACTIC ACID AND BIFIDOBACTERIA ON THE NUMBER OF NATURAL KILLER CELLS IN NORMAL CONDITIONS AND IN CASES OF INTRAVAGINAL STAPHYLOCOCCOSIS

Background. Development of new immunobiotics based on commensal nonpathogenic probiotic bacteria such as lactic acid bacteria and bifidobacteria with antibacterial and immunomodulatory effects is an important area of modern biotechnology. О study to determine the effect of Lactobacillus acidophilus Bifidobacterium animalis VKL and B. animalis VKB (individu-ally) or their different compositions on the number of natural killer cells (NKC) in the spleen of BALB/c mice at normal conditions and in the case of the experimental intravaginal staphylococcosis. Methods . The number of NKC in the spleen was studied using monoclonal phycoerythrin - conjugated antibodies against NKC antigens (MACS, Miltenyi Biotec, Germany). Calculations of NKC as well as analysis of the results were performed using flow cytometry method on a FACStar Plus cytofluorometer. Results. It is shown that the number of NKC in the spleen of intact mice did not change under the influence of L. acidophilus ІМ V В -7279, L. casei ІМ V В -7280, B. animalis VKL or B. animalis VKB (individually). But, using L. acidophilus ІМ V В -7279, L. casei ІМ V В -7280, L. delbrueckii subsp. bulgaricus ІМ V В -7281, B. animalis VKL and B. animalis VKB (individually) or their different compositions for colonization of the vagina in the case of intravaginal staphylococcosis associated with increasing of the number of NKC in spleen in different periods of observation. The number of NKC in the spleen of staphylococcus-infected mice completely normalized after treatment with some probiotic compositions. The probiotic bacteria (individually) only partially normalized the number of NKC in the spleen of staphylococcus-infected mice. Conclusions. Thus, L. acidophilus ІМ V В -7279, L. casei ІМ V В -7280, L. delbrueckii subsp. bulgaricus ІМ V В -7281 or B. animalis VKL (individually) or their various compositions are promising to create highly effective immunobiotics, that are able to increase the innate immunity in cases of infections.


Introduction
Infectious diseases caused by pathogenic or opportunistic bacteria are a vast group of human diseases that are frequently associated with immunosuppression. Recently obtained evidence that in addition to neutrophils and macrophages, natural killer cells (NKC) play an important role in host defense against extracellular bacterial infections [1][2][3]. Numerous experimental and clinical studies of antibacterial effects of activated NKC have demonstrated that they directly kill bacteria using soluble factors and have an indirect effect through interaction with other immune cells such as dendritic cells (DC), macrophages and neutrophils, through the production of cytokines (interleukin (IL)-12, IL-15, IL-18 and interferon (IFN)) [1,4]. At several diseases, such as viral infections [5], atherosclerosis [6], chronic fa-tigue, immune dysfunction syndrome [7], cancer [8] the decrease of NKC cytotoxicity or a reduction in their number were observed that also confirms the importance of regular function of these cells in host defense.
Staphylococcus aureus that remains a common cause of nosocomial bacterial infections and asymptomatically colonize the nasal tract, rectum, mouth, genitals and skin, is often resistant to antibiotics and can cause various diseases, including pneumonia, sepsis, septic arthritis, etc. [9][10][11]. The cellular and molecular mechanisms of anti-staphylococcal host defense are closely associated with innate immune response especially with activity of neutrophils, macrophages and NKC [4,[11][12][13]. Experimental studies have shown that NKC involve in host defense against bacterial lung infection in mice [11] or in rats [13] as well as from arthritis in mice [12], which was induced by S. aureus. Thus, the enhancement of innate immunity, in the first place activation of the neutrophils, macrophages, and NKC is a promising direction of development of new therapeutic approaches for the treatment of patients with staphylococcal infection, especially in the case of infection caused by antibiotic-resistant strains.
The use of probiotics based on commensal nonpathogenic probiotic bacteria such as lactic acid bacteria (LAB) and bifidobacteria with antibacterial and immunomodulatory effects probably is an important part of treatment of patients with infectious diseases, including those induced by extracellular bacteria such as Staphylococcus spp etc. [14,15]. It is known that immunomodulatory effects of probiotics are strainspecific and associated with activation of DC, macrophages, epithelial cells, T regulatory cells, effector lymphocytes, B-lymphocytes and NKC [15]. There is the evidence that commensal bacteria, including LAB, affect the regulation of NKC activity and their ability to product IFN-γ that may depends on the LAB-induced dendritic cells [16,17], this helps them to develop a full range of special functions in the periphery and secondary lymphoid organs. On the one hand, NKC, activated by DC, kill infected or transformed cells in the periphery, and on the other hand, play a key role in Th1 polarization response upon interaction with DC [17]. Bifidobacteria also activated NKC in normal conditions and in case of pathologies [18,19].
As we have shown in animal models [20,21] the probiotic strains Lactobacillus acidophilus IMV B-7279, L. casei IMV B-7280, L. delbrueckii subsp. bulgaricus ІМV В-7281, Bifidobacterium animalis VKL and B. animalis VKB from our collection of the probiotic bacteria have a high level of immunomodulatory properties in normal conditions and in cases of infectious and inflammatory diseases. These probiotic bacteria and their various compositions with different efficacy inhibited the persistence of S. aureus strain 8325-4 in the vagina of staphylococcusinfected BALB/c mice. In the case of the experimental intravaginal staphylococcosis L. casei IMV B-7280, as well as most compositions on the basis of these strains of LAB and bifidobacteria caused a normalization of cellular immunity indicators [21].

Problem statement
This study aimed to investigate if the L.

Materials and methods
Experimental studies were performed on sixweek-old female BALB/c mice, synchronized in their estral cycle. All studies were performed taking into account the rules of the "European Convention for the protection of vertebrate animals used for experimental and other scientific purposes" (Strasbourg, 1986) and in accordance with "General ethical principles of experiments on animals". Mice were kept in standard vivarium conditions at a temperature of 22  1 C, they were provided with the full mixed feed and had free access to automatic water bowls.
The bacterial strains used in the study were: L. acidophilus ІМV В-7279, L. casei IMV B-7280, L. delbrueckii subsp. bulgaricus ІМV В-7281 (deposited in the Depositary of microorganisms of the D.K. Zabolotny Institute of Microbiology and Virology, NAS of Ukraine), B. animalis VKL and B. animalis VKB isolated from content of gut of healthy people in the course of laboratory study of fermented biological materials. The lyophilized in Cuddon Freeze Dryer FD1500 (New Zealand) probiotic bacteria were used in our study. The viability of the LAB and bifidobacteria strains was tested before each experiment by monitoring their growth on the Man-Rogosa-Sharpe (MRS) agar medium or Bifidum-agar medium (respectively) at 37 C for 24-48 h.
S. aureus strain 8325-4 (kindly provided to us by Professor V.S. Zuyeva, N.F. Gamaleya Institute of Epidemiology and Microbiology, Russian Federation) that has a plasmid of resistance to gentamicin was chosen for modelling the intravaginal staphylococcosis in mice. S. aureus strain 8325-4 was grown on selective agar medium for staphylococci (BAIRD-PARKER-Agar, Merck, Germany), which contained gentamicin at a concentration of 15 mg/ml, at 37 C for 24 h. After that bacterial cells were washed twice with sterile phosphate-buffered saline (PBS).
Suspension of the S. aureus 8325-4 in PBS was administered once into vagina of BALB/c mice, in the dose of 510 7 cells per animal. The following clinical manifestations of the infection process were observed in the infected mice: significant increase in whitish mucous secretions of the vagina, elevation of body temperature, inactivity and loss of appetite. Suspension of the probiotic bacteria or their different compositions in PBS was administrated into the vagina of intact and staphylococcus-infected mice 1 day after infection at the dose of 110 6 cells per animal, once a day for 7 days. When two, three or four strains of probiotic bacteria were used as a composition, they were used at the concentration to achieve the total number of bacterial cells  110 6  On the 1 st , 3 rd , 6 th and 9 th days after the probiotic bacteria first administration, spleens were obtained from decapitated mice of all groups who had previously received anesthesia. Leukocytes were extracted from the spleen cell suspension by fractionating cells in ficoll-verohrafin density gradient (  1.077 g/cm 3 ) by centrifuging at 1500 rev/min for 15 min. The cells were then washed twice in the RPMI-1640 culture medium by centrifuging at 1500 rev/min for 10 min. Monoclonal phycoerythrinconjugated antibodies against NKC antigens (MACS, Miltenyi Biotec, Germany) were used for phenotyping of the isolated cells. Calculations of NKC, as well as analysis of the results, were performed using flow cytometry method on a FACStar Plus cytofluorometer (Becton-Dickinson, USA).
All digital data received were processed with the help of the Epi Info software (version 6.0) through analysis of variance. The null hypothesis for the control and experimental comparative groups was checked using Wilcoxon-Mann-Whitney (U) criteria. The differences between the groups were considered statistically meaningful at P  0.05.

Results and discussion
We have established that the effect of probiotic strains of LAB and bifidobacteria on the number of NKC in the spleen of intact and staphylococcus-infected mice was different. The number of NKC in the spleen of intact mice, whose vagina was colonized with L. casei IMV B-7280, L. acidophillus IMV B-7279, B. animalis VKL or B. animalis VKB (individually) remain unchanged throughout the observation period compared with intact mice that did not receive probiotic bacteria (Table 1).
In the spleen of staphylococcus-infected mice the number of NKC changed compared with intact mice, depending on the periods of observation. As shown in Table 2, statistically significant decrease in the number of NKC in the spleen of staphylococcusinfected mice was on the 1 st and 6 th days. The downward trend in the number of NKC in the spleen of staphylococcus-infected mice we observed on the 3 rd day. But, the number of NKC in the spleen of these mice was normalized on the 9 th day.
The data presented here demonstrate a time-dependent immunomodulatory effect of probiotic bacteria that we used for the purposes of vagina colonization in the case of intravaginal staphylococcosis in mice. So, treatment of staphylococcus-infected mice with L. acidophilus ІМV В-7279, L. casei ІМV В-7280, L. delbrueckii subsp. bulgaricus ІМV В-7281 or B. animalis VKL (individually) resulted in increasing the number of NKC in the spleen in different periods of observation compared with staphylococcus-infected mice that did not receive probiotic bacteria (control group) (see Table 2). We observed a slight decrease in the number of NKC in the spleen of staphylococcus-infected mice that received L. acidophilus ІМV В-7279, L. delbrueckii subsp. bulgaricus ІМV В-7281 (on the 6 th day) or L. casei ІМV В-7280 (on the 3 rd day) compared with intact mice. But these changes were incomprehensible. The number of NKC in the spleens of staphylococcus-infected mice treated with B. animalis VKB on the 1 st , 3 rd and 6 th days was the same as in the control group.  Note. Significant differences with the indicators of intact mice are represented by * (P  0.05), while differences with the indicators of staphylococcus-infected mice who did not receive probiotic strains or their composition are represented by  (P  0.05). On the 9 th day the number of NKC in the spleens of these mice was less than in the control mice and even in intact mice.
To answer the question of whether we will have the same time-dependent immunomodulatory effect, if we are going to colonize the vagina of staphylococcus-infected mice by several probiotic strains together, we used different probiotic compositions ( Table 3). As shown by our study, the most effective Among the compositions of the three probiotic strains most effective was L. casei ІМV В-7280 -B. animalis VKL -L. acidophilus ІМV В-7279. After administration of this probiotic composition into staphylococcus-infected mice the increase in the number of NKC in the spleen was observed on the 1 st and 6 th days.
The number of NKC increased in the spleen of staphylococcus-infected mice that received L. casei IMV B-7280 -B. animalis VKB -B. animalis VKL composition on the 3 rd and 6 th days. Treatment of staphylococcus-infected mice with L. casei ІМV В-7280 -B. animalis VKB -L. acidophilus ІМV В-7279 composition resulted in increasing of the NKC number in the spleen of staphylococcus-infected mice on the 3 rd , 6 th and 9 th days. L. acidophilus ІМV В-7279 -B. animalis VKL -B. animalis VKB composition was less effective. The number of NKC in the spleens of these mice was increased only on the 6 th and 9 th days. There was a significant increase in the number of NKC in the spleen of staphylococcus-infected mice that received L. casei ІМV В-7280 -B. animalis VKB -B. animalis VKL -L. acidophilus ІМV В-7279 composition on the 3 rd and 6 th days (see Table 3).
Thus, we established that the number of NKC in the spleen of intact mice did not change under the influence of any probiotic strains that we investigated. But, using L. acidophilus ІМV В-7279, L. casei ІМV В-7280, L. delbrueckii subsp. bulgaricus ІМV В-7281 or B. animalis VKL (individually) or their different compositions for the purposes of vagina colonization in the case of intravaginal staphylococcosis was associated with increasing in the number of NKC in the spleen compared with indicators of staphylococcus-infected mice that did not receive probiotic bacteria or even with intact mice in different periods of observation.
The previous study in our laboratory showed that after L. acidophilus ІМV В-7279, L. casei ІМV В-7280, B. animalis VKL or B. animalis VKB (individually) and their different compositions administration into staphylococcus-infected BALB/c mice the growth of S. aureus in the vagina was inhibited and the number of CD3 + and CD4 + T-cells in the spleen, and CD4 + /CD8 + index, which decreased after staphylococcus infection, were increased [21]. L. acidophilus ІМV В-7279, L. casei ІМV В-7280, L. delbrueckii subsp. bulgaricus ІМV В-7281 induced the IL-12 and IFN-γ production by murine macrophages in vitro [22]. Thus, the use of these probiotic bacteria and their different compositions for the purposes of vagina colonization of staphylococcus-infected mice led to activation of the innate and adoptive immunity.
It should be noted that the effect of probiotic bacteria on the NKC is one of the key mechanism for strengthening of the innate immunity, which plays an important role in host defense against infections. Therefore most researchers have directed their efforts to study the effect of probiotic bacteria on the NKC activity and/or change in their number in normal conditions and in cases of pathologies. It has been found that such probiotic strains of LAB as L. pentosus S-PT84 [23], L. brevis KB290 [24], L. paracasei [25] after oral administration into intact mice significantly increased the activity of NKC. The cytotoxic activity of NKC and production of cytokines in the spleen and blood were increased in immunosuppressed mice which were treated with L. sakei K101 and L. plantarum K55-5 [26]. The number of NKC and their cytotoxicity were significantly increased in tumor-bearing C3H/HeN mice after L. casei Shirota use for treatment [27]. Oral administration of L. plantarum A into tumor-bearing BALB/c mice led to increase of the NKC infiltration into tumor tissue and activation of the effector functions of CD8+ T-cells [28]. L. casei ssp casei significantly increased the cytotoxicity of NKC and up-regulated the production of IFN-γ and IL-12 in the spleen cells culture in invasive ductal carcinoma bearing BALB/c mice [29].
Our results show that in the case of bacterial infections the majority of probiotic compositions used in the present study more effectively affect the number of NKC in the spleen than probiotic bacteria individually. Thus, in the spleen of staphylococcusinfected mice the number of NKC that decreased on the 1 st and 6 th days after mice were infected with S. aureus 8325-4, completely normalized after use for treatment of such probiotic compositions as L. casei ІМV В-7280 -B. animalis VKL, L. acidophilus ІМV В-7279 -B. animalis VKB, B. animalis VKL -B. animalis VKB, L. casei ІМV В-7280 -B. animalis VKL -L. acidophilus ІМV В-7279 and L. casei ІМV В-7280 -B. animalis VKB -B. animalis VKL -L. acidophilus ІМV В-7279. The probiotic bacteria individually only partially normalized the number of NKC in the spleen of staphylococcus-infected mice. Perhaps, this is due to the fact that the probiotic bacteria used in the compositions can enhance the growth of each other after the administration into animals that may increase their ability to colonize vagina and immunomodulatory effects.

Сonclusions
So, our results indicate that L. acidophilus ІМV В-7279, L. casei ІМV В-7280, L. delbrueckii subsp. bulgaricus ІМV В-7281, B. animalis VKB and B. animalis VKL (individually) or their different probiotic compositions are promising to create highly effective immunobiotics, that are able to increase the innate immunity in cases of bacterial infections and, possibly, other pathologies. But in the case of intravaginal staphylococcosis, probiotic bacteria individually were less effective than probiotic compositions. It should be noted that for creation of highly effective immunobiotics consisting of several probiotic bacteria it is important to determine their optimal combination and study their activity in different experimental conditions. However, additional studies should be conducted to ensure that these probiotic strains or their different compositions could be used in treatment or prevention of bacterial infections.