Antagonistic Action of Lactobacilli and Bifidobacteria in Relation to Staphylococcus aureus and Their Influence on the Immune Response in Cases of Intravaginal Staphylococcosis in Mice

The antibacterial activity of Lactobacillus casei IMV B-7280, Lact. acidophilus IMV B-7279, Bifidobacterium longum VK1, and B. bifidum VK2 strains or their various compositions in relation to Staphylococcus aureus in vitro and on models of experimental intravaginal staphylococcosis of mice was determined. It was found that under the influence of these strains and their various compositions, the in vitro growth of Staph. aureus was inhibited, and the number of colonies of Staph. aureus plated from the vagina of infected mice was significantly reduced. The antibacterial activity of these strains separately and in compositions correlated with their ability to improve the performance of the immune response. These strains were the most effective in the following compositions: Lact. casei IMV B-7280—B. longum VK1—B. bifidum VK2. Strains of Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. bifidum VK2, and B. longum VK1 are prospective components of future probiotic drugs efficient in treating staphylococcosis and for immunity correction.


Introduction
Dysfunction of the immune system that emerges as the result of changes in microbial ecology, widespread use of modern chemopreparations of various natures, disruption of the normal microbiota, etc., is one of the major causes of increasingly hostile opportunistic commensal pathogens, with subsequent development of infectious diseases of the urogenital tract, including the anogenital area. The normal biota of the vagina mainly consists of lactobacilli and a smaller number of bifidobacteria, staphylococci, streptococci, coryneforms, enterococci, enterobacteria, etc. [19]. The vaginal microflora is now more frequently viewed as an ''ecosystem'' [18]. The vaginal lactic acid bacteria colonize the mucous membranes, maintain the proper acidity (pH 4.3-4.7), control pathogens that cause urinary tract infections and/or sexually transmitted diseases and may also affect the development of the immune response to causative agents of infectious diseases [18,19]. Therefore, disruption of the normal vaginal microflora, especially due to reduction in the number or activity of lactobacilli [30], frequently causes activation of aggressive forms of opportunistic commensal pathogens, resulting in the development of vaginosis or uncomplicated urinary tract infections, as well as the emergence of other pathological conditions.
It is known that uncomplicated infections of urinary tract and vaginosis are often caused by opportunistic commensal bacteria of the Staphylococcus genus [5,15]. Staphylococcosis usually develops in people with reduced nonspecific immunological resistance, as well as in people who received large doses of immune suppressants, antibiotics, hormones, X-rays, etc. The latter has led to emergence of resistant staphylococci. Frequent regressive uncomplicated urinary tract infection can cause serious diseases, such as nephritis, kidney damage, etc. Longlasting bacterial vaginosis caused by staphylococci is associated with a high risk of development of sexually transmitted infectious diseases, which may increase the risk of late miscarriage [7,14,26].
Therefore, developing alternative nature-derived treatment(s) for patients with uncomplicated urinary tract infections and vaginosis is of the utmost concern. This treatment may include healthy vaginal lactic acid bacteria with expressed antibacterial and immune modulatory properties. There are only a few known strains of lactobacilli that demonstrated a therapeutic effect in cases of urogenital infectious diseases on experimental models and in patients' treatment [8].
We have previously characterized the following strains of lactobacilli and bifidobacteria: Lactobacillus casei IMV B-7280, Lact. acidophilus IMV B-7279, Bifidobacterium longum VK1, and B. bifidum VK2. It was found that these strains had in vitro antagonistic effects in relation to a wide range of pathogenic and opportunistic microorganisms, including causative agents of infectious diseases of the urogenital tract. Furthermore, on the model of intact mice, it was shown that in vivo they effectively induced production of endogenous interferon and activated cells of the phagocytic system, without affecting the production of the pro-inflammatory cytokine tumor necrosis factor-a [27].
The aim of this study was to investigate the antistaphylococcal activity of the Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. longum VK1 and B. bifidum VK2 strains and their compositions on the model of experimental intravaginal staphylococcosis of mice, and determine their influence on innate immunity indicators.

Strains
Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. longum VK1 and B. bifidum VK2 strains were used both individually and in various compositions. These strains were previously selected by us from associated cultures in the course of laboratory study of fermented biological materials. The study was performed using bacteria lyophilized in Cuddon Freeze Dryer FD1500 (New Zealand). Before each experiment, the viability of the probiotic cultures was tested by monitoring their growth on the Man-Rogosa-Sharpe (MRS) agar medium at 37°C for 24-48 h.
Staph. aureus 8325-4 (kindly provided to us by Professor V. S. Zuyeva, N. F. Gamaleya Institute of Epidemiology and Microbiology, Russian Federation) had plasmid-based resistance to gentamicin, allowing it to be separated from other strains of vaginal staphylococcus obtained from the environment through the use of selective media containing this antibiotic.
In Vitro Antagonistic Activity Assays The antagonistic activity of Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. longum VK1 and B. bifidum VK2 strains was determined in vitro in relation to the laboratory collection strains Staph. aureus 209-P, Staph. aureus 43 and Staph. aureus 8325-4 (D. K. Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine).
In the study of antagonistic activity of probiotic cultures, the method of perpendicular strokes on the MRS medium [20] was used. Test cultures of lactobacillus or bifidobacteria strains were collected after 24 h of cultivation. The degree of sensitivity of the test cultures was evaluated according to the size of the zones of growth inhibition: 5-15 mm-low-sensitive, 15-20 mm-moderately sensitive, 30-40 mm-highly sensitive.

Model of Staphylococcal Intravaginal Infection and Treatment of Mice with Lactobacilli and/ or Bifidobacteria
Experimental studies were performed on six-week-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. Staphylococcosis was modeled through intravaginal administration of the Staph. aureus 8325-4 daily culture to mice, in doses of 5 9 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.
Twenty-four hours after infection, mice were given an intravaginal injection of a suspension of lyophilized lactobacillus and/or bifidobacteria cells in saline solution at a dose of 1 9 10 6 cells per animal, once per day for 7 days. Strains were injected individually and in the following combinations: acidophilus IMV B-7279; Lact. casei IMV B-7280-B. bifidum VK2-B. longum VK1-Lact. acidophilus IMV B-7279 in equal proportion. A separate group was formed by the infected mice that did not receive these strains or their combinations, but received the saline intravaginally. The control group included intact mice.

Quantification of the Antibacterial Activity In Vivo
On the first, third, sixth, ninth and twelfth day after the injection of the lactobacillus and/or bifidobacteria strains, alone or in various combinations, into the mice, Staph. aureus 8325-4 was collected from the vagina and plated onto a selective medium for staphylococci containing gentamicin. The material was collected using standardized sterile cotton tampons. Swabs from each tampon were performed with 1 ml of saline. After cultivation at 37°C for 24 h, the number of colony forming units was counted, given that one such colony corresponds to one bacterium.

Determining the Number of T-and B-Lymphocytes in the Spleen
On the first, third, sixth and ninth day after the injection of the lactobacillus and/or bifidobacteria strains, alone or in combinations, into the mice, the spleens were extracted from the killed mice, and suspensions of splenocytes were prepared in RPMI-1640 culture medium. Leukocytes were extracted from the spleen cell suspension by fractionating cells in ficoll-verohrafin density gradient (q = 1.077 g/cm 3 ) by centrifuging (on the centrifuge/vortex Multi-Spin MSC-3000) at 400g for 15 min. The cells were then washed twice in the RPMI-1640 culture medium by centrifuging at 400g for 10 min. Surface antigens of T-and B-lymphocytes were investigated with the help of the direct immunofluorescence method. Monoclonal antibodies to CD3?, CD4?, CD8? and CD19? antigens (MACS, Miltenyi Biotec, Germany) were used in the work. Calculation of T-and B-lymphocytes and analysis of the results were performed on a FACStar Plus cytofluorometer (Becton-Dickinson, USA).

Statistics
All digital data received were processed with the help of the Origin Pro 8.5. software through analysis of variance. Numerical data were represented as arithmetic average and standard error (M ± m). The null hypothesis for the control and experimental comparative groups was checked using Wilcoxon-Mann-Whitney (U) and Kolmogorov-Smirnov nonparametric criteria. The differences between the groups were considered statistically meaningful at P \ 0.05. The following data were obtained from the study of the anti-staphylococcal activity of the probiotic strains in vivo. It was found that Staph. aureus 8325-4 was recovered from the vagina of infected mice who did not receive probiotic cultures or their compositions during the entire period of observation (days 1-12) ( Fig. 1). At the same time, after injection of probiotic cultures in monocultures or in various compositions into the infected mice, the number of colonies of Staph. aureus 8325-4 decreased significantly compared to the infected mice that did not receive these strains or their compositions. Thus, after injection of Lact. acidophilus IMV B-7279, Lact. casei IMV B-7280, B. longum VK1 or B. bifidum VK2 separately to mice, a decrease in the number of Staph. aureus 8325-4 colonies, which was recovered from the vagina, was observed from the first day and throughout the entire subsequent period of observation. However, if we compare these probiotic cultures on an individual basis ( acidophilus IMV B-7279 compositions separately into the mice, staphylococcus was recovered from the vagina on the twelfth day, but at a much smaller amount than that from the infected mice who did not receive compositions of probiotic cultures. It is worth noting that, contrary to other compositions of the two strains, it was the Lact. casei IMV B-7280-B. longum VK1 composition that was a more active antagonist of  Analyzing the data obtained, it is possible to conclude that the Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 composition appeared to be the biggest antagonist of Staph. aureus 8325-4. The anti-staphylococcal activity of the Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 composition on the first day after injection into the mice was the same as after injecting these monocultures separately. However, on the third, sixth, and ninth days, the anti-staphylococcal activity of this composition appeared to be better than that of the monocultures injected separately. On the twelfth day, Staph. aureus 8325-4 was still recovered out from the vagina of infected mice who received B. longum VK1 separately. On the first day, the efficiency of the Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 composition was lower than that of the Lact. casei IMV B-7280-B. longum VK1 and Lact. casei IMV B-7280-B. bifidum VK2 compositions, but on the third and sixth days it was the same as that of the compositions of two strains. At the same time, on the ninth day, staphylococcus was fully eliminated from the vagina of the mice that received the Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 composition, but was still recovered after the injection of the Lact. casei  (Table 2). After infection of the mice with staphylococcus on the third day, a reduction in the number After the injection of some individual probiotic cultures and some of their compositions into the infected mice, an increase in the level of indicators controlling the number of CD3? and/or CD4? cells (third day) in the spleen was observed, while the level of CD8? cells was normal, and an increase in the number of CD19? cells was observed in different periods. It was found ( Table 3) that the number of CD3? and CD4? cells on the third day in the spleens of infected mice increased under the influence of Lact. casei IMV B-7280 compared with the infected mice that did not receive the probiotic cultures. Following injection of B. longum VK1 into the infected mice, on the third day, only a tendency to increase in the number of CD3? cells and a probable increase in the number of CD4? cells was observed. The number of CD3? and CD4? cells was smaller on the third day in the spleen of infected mice who received either Lact. acidophilus IMV B-7279 or B. bifidum VK2 separately than in the control. After the injection of Lact. acidophilus IMV B-7279 into the infected mice, the number of CD3? cells on the first day appeared to be lower than in the control. However, it should be noted that after the injection of all these probiotic cultures into the infected mice, a separate increase in the CD4/CD8 index was detected on the third day compared with the infected mice who did not receive probiotic cultures. This may be due to an increase in the number of CD4? cells under the influence of Lact. casei IMV B-7280 or B. longum VK1, or due to the reduction in the number of CD8? cells under the influence of Lact. acidophilus IMV B-7279 or B. bifidum  casei IMV B-7280 composition, the number of these cells also appeared to be low on the first day compared with both the control and the infected mice that did not receive the composition of probiotic cultures. The CD4/CD8 index rose on the third day following the injection of the Lact. casei IMV B-7280-B. bifidum VK2 composition into the infected mice (due to an increase in the number of CD4? cells) and also on the sixth and ninth days (due to the tendency to  VK1-B. bifidum VK2 composition into the infected mice, this indicator also increased on the first day. Only on the third day, a tendency of the CD4/CD8 index to increase was detected under the influence of the all studied groups, but for the Lact. acidophilus-B. longum VK1-B. bifidum VK2 composition, this indicator rose on the first day. It was shown that the number of CD19? cells (Table 4) [31], which is a frequent cause of dysbiosis and of development of uncomplicated infections of the human urogenital tract. It is considered that the use of probiotics based on lactobacilli and bifidobacteria in the treatment of such patients is an alternative therapy to antibiotics and chemotherapy [8]. Studies on the antagonistic interactions between lactic acid bacteria and Staph. aureus have been carried out in various laboratories throughout the world over the past several decades [2]. It has been repeatedly shown that many strains of lactobacilli and bifidobacteria taken from the intestines and vagina We studied changes in the numbers of T-and B-lymphocytes, and certain subpopulations of T-lymphocytes in the spleen of the mice infected with Staph. aureus 8325-4. Experimental models and studies on patients have shown that the immunomodulatory mechanism of action of many probiotic strains of lactobacilli and bifidobacteria involves activation of cellular and humoral immunity: the numbers of T-and B-lymphocytes increases, as does their proliferative activity and production of a series of imunoregulatory cytokines [3,10,13,32,33].

Antagonistic
As we previously established on the model of intact mice, Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. longum VK1, and B. bifidum VK2 intensified the production of endogenous interferon and activated macrophages [27]. In this study, we showed that under the influence of some strains, particularly Lact. casei IMV B-7280 or B. longum VK1 alone, or several of the compositions of multiple probiotic cultures, the number of CD3? and/or CD4? cells in the spleen of the infected mice increased to the level of control on the third day, compared with the infected mice who did not receive probiotic cultures or their compositions. After injection of the Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 composition (which demonstrated the best antistaphylococcal effect in vivo) into the mice infected with staphylococcus, an increase in the number of CD4? cells was observed in the spleen on the third day, while the number of CD3? cells was the same as in the infected mice who did not receive probiotic cultures or their compositions. Moreover, the number of CD19? cells increased on the ninth day in comparison with the control indicators (intact mice). Combining Lact. casei IMV B-7280 and B. longum VK1 in one composition is successful due to normalization of the number of CD3? and CD4? cells observed in the spleens of the infected mice. In terms of their individual immune modulatory activities, the first strain induces the ''late'' interferon, while the second strain induces the ''early'' interferon, as we previously showed [18] on the model of intact mice. Thus, in our studies, there was a correlation between the ability of strains of lactobacilli and bifidobacteria or their compositions to inhibit the growth of Staph. aureus in vivo and their various immune modulatory properties. However, it should be noted that the immunomodulatory action of Lact. casei IMV B-7280 and B. longum VK1 separately, as well as some compositions of other strains, was better than that of the Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 composition having the highest anti-staphylococcal action in vivo. The probiotic strains studied by us, individually and in various compositions, may use different mechanisms of influence on the development of the immune response.
Thus, Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. longum VK1, or B. bifidum VK2 can be used for creating probiotic drugs effective against Staph. aureus and having immunomodulatory effect. Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2 appeared to be the most promising composition. Before we can create a commercial probiotic drug on the basis of these strains of probiotic cultures for intravaginal use, further research must be conducted. We plan to determine the strains' influence on the growth of opportunistic flora, especially fungi of the Candida genus, as well as on the balance of the production of pro-and anti-inflammatory cytokines, namely the Th1-and Th2-types of cytokines. Our results confirm the validity of the requirements of the European regulatory legislation in the field of probiotics regarding the need for comprehensive studies of biological activity of both separate cultures and their combinations, which would allow for the creation of effective probiotic drugs based on monocultures of lactobacilli and/or bifidobacteria or their various combinations.
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