Biofilm formation of Candida Spp. isolated from the vagina and antibiofilm activities of lactic acid bacteria on the these Candida Isolates

Abstract Background In this study, it was aimed to investigate the effects of bacterial cells and cell-free filtrates of Lactobacillus acidophilus 8MR7 and Lactobacillus paracasei subspecies paracasei 10MR8 on the biofilm formation of 3 Candida tropicalis, 3 C. glabrata and 12 C. albicans isolated from the vagina and identified their virulence factors. Methods Haemolytic activities esterase activities, and phospholipase activities as virulence factors of Candida strains were determined. Biofilm formations of these isolates were determined by Congo Red agar and microtitration plate method. Antibiofilm activities of bacterial cells and cell-free filtrates of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 on Candida isolates were determined by the microtitration plate method. Result Bacterial cells of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 were not very effective in the inhibition of biofilm, whereas it has been observed that the cell-free filtrates of these bacteria inhibit the formation of biofilms of Candida strains. Although the main mechanism for inhibiting the formation of Candida spp. biofilm is the competition for adhesion, it is concluded that the substances contained in the cell-free filtrates of lactic acid bacteria are also important. Conclusion These isolates promise hope as potential bacteria that can be used for anti-adhesion purposes in health-care materials.


Introduction
The human microbiome colonized in the human body are composed of numerous microorganisms. Different microbial communities have been located in the vagina, mouth, skin, gastrointestinal tract, nose, urethra and other parts of the body 1 . Besides, the Candida species are also found in the normal microbiome of human especially they usually colonize on the skin and mucous membranes. Besides, Candida species are one of the most common pathogens in humans. They cause a wide spectrum of disease ranging from non-invasive superficial infections to infections involving the deep tissues.
Biofilm is a collection of microorganisms that are embedded in the exopolysaccharide matrix and are irreversibly attached to each other and to a surface. The structure and composition of Candida spp. biofilm can vary according to various environmental conditions. This reduces the success of the treatment. Several proposals have been made to prevent this in biofilm-forming Candida species isolates.
Lactobacilli are dominant in the vaginal microbiome of a healthy woman 2 . Lactobacilli play an important role in the protection of normal vaginal microbiome, inhibiting development of pathogenic and opportunistic organisms 3 . They have the properties such as tolerance to acid and bile salts, adhesion to the human intestinal mucosa, temporary colonization of the human gastro-intestinal tract, production of antimicrobial agents 4 . Lactic acid bacteria inhibit development of pathogen microorganisms by producing organic acids such as lactic acid, hydrogen peroxide, bacteriocin or bacteriocin-like substances. They compete with pathogens for food and colonization. Besides, these bacteria also have benefits such as stimulation of the immune system, lowering of serum cholesterol level, and reduction of cancer risk. Recently, several important biological functions of some lactic acid bacteria such as anti-aging and anti-oxidant activities have been revealed 5 . In the present study, it was determined the biofilm formations in vitro conditions of 18 Candida spp. isolates isolated from the vagina and investigate the inhibition effects of Lactobacillus acidophilus 8MR7 and Lactobacillus paracasei subspecies paracasei 10MR8 on the biofilm formation of Candida spp.

Materials and methods Materials Microorganisms
In this study, Lactobacillus acidophilus 8MR7 and L. paracesei subspecies 10MR8 and 18 Candida spp. (3 C. tropicalis, 3 C. glabrata, 12 C. albicans) that isolated from the vagina of healthy women and identified by the API-CHL 50 test and the MALDI-TOF Mass Spectrometry Technique in another study were used. Bacteria and Candida isolates were incubated on the de Man, Rogosa, and Sharpe (MRS) agar and Sabouraud dextrose agar (SDA) at 37°C 5% CO2 for 48 h and at 37°C for 48 h, respectively.

Determination of hemolytic activity
For the determination of hemolytic activity, the Candida spp. isolates were incubated on the sheep blood agar at 37°C for 48 h. Following incubation, beta hemolytic activity around the colony was determined by the existence of light-transmitting transparent zone, and alpha hemolytic activity was determined by the presence of dark green reproduction 6 .

Determination of esterase activity
Candida isolates were allowed to incubate for 48 h at 37 ° C in Sabouraud dextrose broth (SDB). After the concentrations of Candida isolates were adjusted to 10 7 cfu / mL in 0.85% physiological saline following the incubation, 5 μL of each culture was added dropwise to each culture on tween 80 agar medium. The petri dishes were incubated at 37 ° C for 10 days and after the incubation, the zone formation around the colony was examined. The experiment was performed in duplicate manner 7 .

Determination of antifungal activities of lactic acid bacteria
Antifungal activity against Candida spp. isolates of L. acidophilus 8MR7 and L. paracesei subspecies paracasei 10MR8 was determined by the duplicate agar method. 5 μL (10 8 cfu / mL) of lactic acid bacteria culture was incubated dropwise on MRS agar (de Man, Rogosa, and Sharpe agar) at 35 ° C 5% CO2 for 48 h. After incubation, Candida spp. isolates (105cfu / mL) growing in the SDB was inoculated with Sabouraud dextrose semi-solid agar. After the semi-solid agar was thoroughly mixed, 7 mL of the solution was poured slowly onto the surface of the petri dishes containing the lactic acid bacteria. The petri dishes were allowed to incubate at 37 ° C for 48 h. After incubation, the zone diameters around the lactic acid bacteria were measured and re-corded9.

Determination of biofilm formation
The experiment was carried out in duplicate manner. The biofilm formation of the isolates was determined on Congo red agar and by the microtitration plate method.
Determination of biofilm formation in Congo red agar Candida spp. cultures were incubated in the Congo red agar (CRA) at 35 ° C for 48 h. The isolates forming black colonies were assessed as forming biofilm 10 .

Determination of biofilm formation by microtitration plate method
After the Candida spp. isolates produced in the SDB were adjusted to be 10 7 cfu / mL, they were distributed as 20 μL in each well of the 96-well plate. 180 μL synthetic dextrose liquid (SDL) medium containing 2.5% glucose was transferred onto it and incubated for 48 h at 35 ° C. After incubation, the plates were emptied and each well was washed 3 times with sterile physiological saline. The wells were fixed with 200μL 99% methanol for 15 minutes. At the end of this period, the wells were emptied and left to dry. Subsequently, each well was stained with 200 μL 2% crystal violet for 5 minutes. When this period ended, the wells were washed with distilled water and dried. After the drying, the wells were treated with 160μL 33% glacial acetic acid and assessed spectrophotometrically at 570 nm. According to the optical density (OD), biofilm formation was evaluated. If the OD values were 0 ≤ OD570 ≤ 0.120 (-), 0.121 ≤ OD570 ≤ 0.240 (+), 0.241 ≤ OD570 ≤ 0.500 (++), OD570 ≥ 0.500 (+++), the biofilm was interpreted as negative, weak, intermediate, strong, respectively 11 . The test was carried out in duplicate manner.

Preparation of cell-free filtrate
Lactic acid bacteria isolates were incubated in MRS broth at 35 ° C 5% CO2 for 48 h. After incubation, the cultures were centrifuged at 10.000 rpm for 10 minutes at 4 ° C, supernatant was filtered through a 0.2 μm filter.

Determination of the effects of bacterial cells and cell-free filtrate of lactic acid bacteria on the biofilm formation of Candida spp.
For antibiofilm activity of bacterial cells of lactic acid bacteria, 10 μL of the Candida spp. isolates (10 7 cfu / mL) produced in the SDB were distributed into the a 96 -wells plate. 90 μL SDB containing 2.5% glucose was transferred onto it. These wells were incubated at 35 ° C for 48 h by adding 10 μL of bacterial cells of lactic acid bacteria (109 cfu / mL) cultured in the MRS broth and 90 μL MRS broth containing of 2.5% glucose were distributed into the plate. For antibiofilm activity of cellfree filtrate of lactic acid bacteria, 10 μL of Candida spp. isolates (107 cfu / mL) cultured in the SDB were distributed into the wells. 140 μL SDL containing 2.5% glucose was transferred onto it. Cell-free filtrate of lactic acid bacteria was added at 50 μL and incubated at 35 ° C for 48 h. Candida and SDB were added to the wells as control. The amount of biofilm was determined according to the microtitration plate method given above. Table I illustrates the hemolytic activity results of yeast isolates isolated from the vagina. Hemolysis was observed in 9 out of 18 Candida strains. In addition, the isolates of C. albicans 24P1, C. albicans 25P1, C. albicans 5MR2, C. albicans 14P1, C. albicans 19P3, C. albicans 27P2, C. tropicalis 1Ç1, C. glabrata 17P2, and C. glabrata 16P did not generate inhibition zone (Table I). While no biofilm formation was observed in C.albicans 19P3 and C. tropicalis 1C3 by the microtitration plate method, high biofilm formation was observed on the Congo red agar. Conversely, high levels of biofilm formation were observed by microtitration plate method in C. tropicalis 1C1, but it was found there was no biofilm formation on the Congo red agar (Table I).
It was observed that the effect of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 isolates on the biofilm formation of Candida isolates varied according to the isolates. L. acidophilus 8MR7 generally increased the biofilm formation. Biofilm formation in C. albicans 15P, C. albicans 24P1, and C. albicans 14 P1 isolates was recorded lower in comparison to the control. It was observed that although bacterial cells of L. paracesei subspecies paracasei 10MR8 isolate reduced the biofilm formation in other Candida spp. isolates except C. albicans 15P, C. albicans 13P2 C. tropicalis 29P1, and C. tropicalis 1C3 (Figure I), cell-free filtrates of the lactic acid bacteria were more effective on all the Candida spp. isolates ( Figure II). Cell-free filtrate of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 isolates caused a significant decrease on the biofilm of the Candida spp. isolates.

Discussion
The importance of health-friendly bacteria and their products is increasing day by day. In our study, in order to determine the virulence factors of yeast isolates isolated from the vagina, we investigated the hemolysis, esterase, phospholipase activity and biofilm of the isolates. According to the results of hemolytic activity of yeast isolates, hemolysis was observed in nine isolates (C. albicans 18P1, C. albicans 30P, C. albicans 15P, C. albicans 25P1, C. albicans 24P2, C. albicans 14P1, C. albicans 27P2, C. glabrata 17P2, and C. glabrata 16P). In a study conducted by Luo et al., it was revealed that C. albicans, C. glabrata, and C. tropicalis isolates isolated from human had alpha hemolytic activity6. In another study, it was reported that 4 of the 63 C. albicans isolates had alpha hemolysis, 53 had alpha + beta hemolysis, 6 did not have hemolysis and 2 C. tropicalis isolates did not have hemolytic activity12. All of our Candida spp. isolates had high phospholipase activity. It was found that while 7 of the C. albicans strains (C. albicans 18P1, C. albicans 30P, C. albicans 15P, C. albicans 24P2, C. albicans 13P1, C. albicans 18MR11, and C. albicans 13P2), and two of the C. tropicalis strains (C. tropicalis 1Ç3, C. tropicalis 1Ç1) had esterase activity, none of C. glabrata strains had esterase activity. It was revealed that most of the pathogenic Candida species released some lipolytic enzymes such as esterase and phospholipases 7 . It was also reported that phospholipases were highly likely to increase the pathogenicity of C. albicans. Gültekin et al. 13 reported that C. albicans strains had phospholipase activity, but this activity was not available in non-albicans species. Phospholipase activity was detected in 73% of vaginal discharge samples 13 . Gültekin et al. 14 reported that none of the 65 vaginal isolates (C. glabrata) had esterase and phospholipase activity; only two of the isolates produced biofilm 14 .
The formation of biofilm was changed according to the strains. It was observed that there were some differences between the two methods. However, it was found that biofilm formation was generally high. Cevahir  Fracchia et al. and Zakaria reported that the lactic acid bacteria producing bio surfactants had high anti-adhesion capacity against the pathogenic C. albicans 24,25 . The researcher suggested that the anti-adhesion properties of lactic acid bacteria might be due to the bio surfactant in the filtrate. Similar findings were also emphasized by Gudiña et al. Zeraik et al. reported that the anti-adhesion effect of lactic acid bacteria filtrate varied according to the properties of supernatant, test microorganism and surface properties 22,26 . They report that when the surface was covered with filtrate containing biosurfactant, the surface became hydrophilic and reduced microbial adhesion 26,27 . In our study, the fact that supernatants of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 were more effective might be to do with bio surfactant production.
The inhibition of surface adhesion of pathogenic bacteria to filtrates of the lactic acid bacteria is of great importance for health. This may also be important for biomedical instruments. Falagas and Makris emphasized that the bio surfactants isolated from Lactobacillus might play an important role in inhibiting adhesion in the maintenance equipment such as catheters and other medical devices used in hospitals 28 .

Conclusion
While the main mechanism of lactic acid bacteria to inhibit biofilm formation of Candida species is the competition in the adhesion zone, the materials contained in the cell-free filtrates of lactic acid bacteria are important as well. When we look at the cell-free filtrates of L. acidophilus 8MR7 and L. paracasei subspecies paracasei 10MR8 isolates, it is seen that they have a significant anti-adhesion activity. These isolates are promising as potential bacteria that can be used for anti-adhesion purposes in materials used in healthcare, as well as especially as the support for the health of the vagina.
ty Non-Interventional Clinical Researches Ethics Board on 11/04/2013 with decision number38. All applicable international, national, and/ or institutional guidelines for the care and use of human were followed.