The antimicrobial effects of selenium nanoparticle-enriched probiotics and their fermented broth against Candida albicans

Background Lactic acid bacteria are considered important probiotics for prevention of some infections. The aim of this work was to investigate the effect of selenium dioxide on the antifungal activity of Lactobacillus plantarum and L. johnsonii against Candida albicans. Methods Lactobacillus plantarum and L. johnsonii cells, grown in the presence and absence of selenium dioxide, and their cell-free spent culture media were tested for antifungal activity against C. albicans ATCC 14053 by a hole-plate diffusion method and a time-kill assay. Results Both L. plantarum and L. johnsonii reduced selenium dioxide to cell-associated elemental selenium nanoparticles. The cell-free spent culture media, from both Lactobacillus species that had been grown with selenium dioxide for 48 h, showed enhanced antifungal activity against C. albicans. Enhanced antifungal activity of cell biomass against C. albicans was also observed in cultures grown with selenium dioxide. Conclusions Selenium dioxide-treated Lactobacillus spp. or their cell-free spent broth inhibited the growth of C. albicans and should be investigated for possible use in anti-Candida probiotic formulations in future.


Introduction
Candida albicans, although it is a commensal yeast in the oral cavity, gastrointestinal tract and urogenital tract, can cause a variety of mild to serious infections. C. albicans usually infects immunocompromised patients or others who use antibiotics for a long time [1]. One reason for the overgrowth of C. albicans and infection is disequilibrium in the microbiota [2,3]. Probiotics are microorganisms which, when consumed in adequate amounts, can improve intestinal microbial balance and provide benefits for human health [4]. Lactic acid bacteria (LAB) are important probiotics and also part of the normal Gram-positive microflora inhabiting the intestinal mucosa. They aid in prevention of colonization by pathogenic microorganisms [5]. In the vagina, normal Lactobacillus species have a critical role in protection against vaginal infections and the transmission of pathogens responsible for sexually transmitted diseases [4][5][6][7][8]. These bacteria produce lactic acid, acetic acid, hydrogen peroxide, and other antimicrobial substances, which allow them to prevent the colonization of pathogens [8,9]. Some LAB strains can protect the human vagina from candidiasis through the production of these exometabolites [10][11][12][13][14]. As yet uncharacterized metabolites from seleniumenriched probiotics have recently been shown to exert an antibacterial effect against Escherichia coli [15]. In the present work, we aimed to study the antimicrobial effect of two selenium-enriched Lactobacillus spp. cultures and their exometabolites against C. albicans ATCC 14053 and to compare these results with anti-Candida effects of spent broth of non-selenium-enriched Lactobacillus cultures.

Microbial strains
L. plantarum (ATCC 8014) and C. albicans (ATCC 14053) were obtained from the American Type Culture Collection (ATCC). The other Lactobacillus was a clinical isolate, which was identified as L. johnsonii during a previous study [16].

Effect of Lactobacillus species on selenium dioxide
One hundred milliliters of DeMan-Rogosa-Sharpe (MRS) broth (Merck, Darmstadt, Germany) was used for inoculation of L. plantarum and L. johnsonii strains. The cultures were grown at 37°C in a shaker incubator for 24 h. After this time, selenium dioxide (Merck Schuchardt, Hohenbrunn, Germany) was dissolved in distilled water (289.5 mg/l) and sterilized by a Millipore filter apparatus (Millipore Corporation, Milford, MA, USA). This selenium dioxide solution was added aseptically to each of the Lactobacillus cultures to obtain a final concentration of 200 mg/l of Se. The cultures were further incubated at 37°C for 96 h. Two-milliliter samples were withdrawn at zero time and at intervals (24, 48, and 96 h) under aseptic conditions. The bacterial cells were removed from the cultures by centrifugation at 5,000 × g for 10 min (Hettich Mikro 200, Tuttlingen, Germany). The supernatant at each time interval was used to measure the concentration of Se remaining in the medium by Somer and Kutay's spectrophotometric method [17]. The cell pellet from a culture of L. plantarum and isolated selenium nanoparticles (SeNPs) were examined at 100 kV by a Philips EM-208 transmission electron microscope (TEM) (FEI Ltd., Eindhoven, The Netherlands) to evaluate the presence and the size of Se NPs deposited inside the L. plantarum cells as previously described [18]. To determine the elemental composition of the nanoparticles (NPs), energy dispersive X-ray spectrum (EDX) microanalysis (Vega Tescan, Brno, Czech Republic) was also performed.

Preparation of Lactobacillus cultures for antifungal activity assays
Four flasks, each containing 100 ml MRS broth, were used for inoculation of two sets each of L. plantarum and L. johnsonii strains. The cultures were aerobically grown at 37°C in a shaker incubator for 24 h and one set of each bacterium was treated with selenium dioxide as previously described [16]. The cultures were incubated at 37°C for another 96 h. At zero time and every 24 h, 1 ml samples from all four sets of cultures were harvested under aseptic conditions. The samples were centrifuged at 5000 × g for 15 min. All collected supernatants were assayed for antifungal activity against C. albicans. Cultures incubated for additional time (96 h) were subjected to further centrifugations to isolate enough culture supernatants for the time-kill assay.
Assay for antifungal activity of L. plantarum and L. johnsonii grown with or without selenium dioxide Both a conventional hole-plate diffusion method and a time-kill assay were used to detect antimicrobial activity in the samples. The supernatants of L. plantarum and L. johnsonii, grown with or without selenium dioxide, were sterilized by filtration through a 0.22 μm Millipore filter. Sabouraud dextrose agar (SDA) plates were inoculated with C. albicans and used to test anti-Candida effects of the collected supernatants from each culture. 14-mm diameter holes were punched aseptically in each plate and filled with 100 μl of the cell-free supernatants. As a negative control, sterile MRS liquid medium (100 μl) was used. The plates were incubated aerobically for 18 h at 37°C and the diameters of the inhibition zones (mm) were measured.
The effect of cell-free supernatants of 72-h cultures of L. plantarum and L. johnsonii, grown with or without selenium dioxide, on the survival of C. albicans was also evaluated by conventional time-kill assays. To the tubes containing each of the supernatants, C. albicans (3 × 10 6 /ml) was added and incubated at 37°C. The viability of C. albicans was studied by plating samples taken at different intervals (0.5, 4, 8, 12, and 24 h) on SDA medium and counting the CFU of surviving C. albicans.
The antifungal activity of bacterial cells of L. plantarum and L. johnsonii, grown with or without selenium dioxide, was also assayed with C. albicans. Each of the bacterial pellets prepared as previously described was suspended in a normal saline solution containing 3 × 10 6 CFU/ml of C. albicans. The yeast: bacterial ratio in the suspension was approximately 1/1000 CFU/ml. Samples were withdrawn at different intervals (0.5, 4, 8, 12, and 24 h) for determining the number of surviving C. albicans in each challenge test. The samples were plated on SDA medium and incubated at 37°C overnight. The surviving C. albicans CFU were counted. These experiments were repeated three times.

Selenium dioxide reduction
After 24 h incubation with both L. plantarum and L. johnsonii, the concentrations of selenium in the supernatants were considerably reduced. Figure 1  cultures ( Figure 1). No appreciable amount of Se was present in the culture supernatant of any of the strains after 96 h of incubation. The selenium-enriched L. plantarum cells were selected for a TEM experiment (Figure 2A). Spherical SeNPs of various sizes had formed inside the L. plantarum cells ( Figure 2B). Figure 2C shows a size histogram of the SeNPs inside the cells; the particle sizes ranged from 25 to 250 nm. Furthermore, EDX microanalysis of the separated NPs exhibited Se absorption peaks consisting of SeLα, SeKα and SeKβ at 1.37, 11.22 and 12.49 keV, respectively ( Figure 2D) and confirmed the presence of Se in the samples.
Anti-Candida effect of fermented broth of L. plantarum and L. johnsonii The anti-Candida effects of the supernatants from L. plantarum and L. johnsonii, grown with and without selenium dioxide, were compared by measuring the zone of inhibition formed around each of the samples applied to the plates in the hole-plate diffusion method (Table 1). Negligible inhibition zones were observed on the plates containing culture supernatants of either of the species grown without selenium dioxide or sterile MRS broth supplemented with selenium dioxide (200 mg/l). Culture supernatants from L. plantarum and L. johnsonii grown with selenium dioxide for 48, 72, and 96 h showed potent anti-Candida activity and inhibited growth. The maximum antifungal activity was observed in 72 h cultures ( Table 1). The time-kill assay, measuring the effect of culture supernatants of each strain, grown with or without selenium dioxide, on the viability of C. albicans, confirmed the increase in the antifungal activity of the strains grown with selenium dioxide (Figure 3). Whereas incubation of C. albicans with the supernatants of all cultures decreased viability, the number of surviving C. albicans cells substantially decreased when incubated with the supernatants of cultures grown for 72 h with selenium dioxide. The difference in the effect, which was observed even after 0.5 h incubation, increased with time. No viable C. albicans was present after 4 h incubation with culture supernatants of either species grown with selenium dioxide, but some viable C. albicans cells were present even after 24 h incubation with culture supernatants of species grown without selenium dioxide.
Antifungal effect of L. plantarum and L. johnsonii cells against C. albicans The anti-Candida effects of the selenium-enriched and non-enriched cells of L. plantarum and L. johnsonii were also evaluated. The viability of C. albicans decreased following co-culture with both bacteria (Figure 4), but cells from selenium-enriched cultures were more effective at killing C. albicans (Figure 4). After 0.5 h incubation of C. albicans with the Lactobacillus strains grown without selenium dioxide, viability of C. albicans had decreased by approximately 10-fold, whereas a 1000-fold decrease was seen when incubated with the SeNPs-enriched species ( Figure 4). However, the number of viable cells did not decrease with increased incubation time.

Discussion
Inhibition of C. albicans by some strains of Lactobacillus species is known and results of clinical trials have shown the effectiveness of some strains of Lactobacillus spp. in prevention of C. albicans infections [19]. In this study, we have evaluated the interaction of L. plantarum and L. johnsonii with selenium dioxide on the antifungal activity of these bacteria for C. albicans. Both strains converted selenium dioxide to SeNPs of various sizes, which accumulated inside the cells. Whereas both cells and culture supernatants had anti-C. albicans activity, substantially higher antifungal activity was observed in the culture supernatants of strains grown with selenium dioxide. It appears that selenium dioxide in the cultures enhanced production of soluble metabolites involved in killing C. albicans cells.
The antifungal activity of L. plantarum is related to the production of specific compounds, such as phenyllactic acid and 4-hydrophenyllactic acid [20]. When C. albicans was mixed with the SeNPs enriched Lactobacillus cells or the cell-free fermented broth, clear decreases in viability were observed. The addition of selenium dioxide to the culture medium of either Lactobacillus species led to potent increases in antifungal activity against C. albicans.
Other studies indicate that the adverse effect of LAB can result from production of lactic acid, acetic acid, H 2 O 2 , CO 2 , bacteriocins, and uncharacterized compounds [10][11][12][13]. Therefore, selenium dioxide may induce the production of these exometabolites or induce the synthesis of novel anti-Candida compounds. At this time, the nature of the exometabolites responsible for the observed antimicrobial activity is not known, and further bioassay- Table 1 The antimicrobial activity of spent broth of L. johnsonii and L. plantarum cultures grown with or without selenium dioxide against C. albicans, as measured by the diameter of the zone of inhibition formed in the hole-plate diffusion assay guided fractionation assays should be used to isolate and characterize the active constituent(s). During the cultivation of Lactobacillus spp. in MRS broth containing selenium dioxide, this compound was reduced to elemental SeNPs, which accumulated in intracellular spaces of Lactobacillus spp. and may have contributed to the increased antifungal activity of the treated cells.
The modification of the microenvironment of LAB is being considered as a means of preventing infections of the urogenital and intestinal tracts [6][7][8]. The application of SeNPs enriched-Lactobacillus may be a good approach for the design of new strategies in enhancing the activities of probiotics for curing infections caused by urogenital pathogens, such as C. albicans. species or their cell-free culture media. A greater decrease in viability of C. albicans was seen for bacteria grown with selenium dioxide than for non-Se-enriched bacteria. A direct antifungal effect was observed when SeNPs-enriched Lactobacillus spp. were co-cultured with C. albicans. In addition, evidence for release of potent exometabolites was indicated by the strong inhibition of growth of C. albicans treated with cell-free culture media from the SeNPs-enriched Lactobacillus species. This is the first time in which antifungal activity of the combination of Lactobacillus spp. and selenium has been studied. This phenomenon should be further evaluated for its practical application.