An in vitro study on the antifungal and antibiofilm activities of probiotic bacteria against Candida species isolated from orthodontic appliances and dental caries

Candida species are opportunistic pathogens that may cause infections in predisposed persons. This work aimed to detect the antifungal and antibiofilm potentials of Lactobacillus acidophilus and Lactobacillus plantarum supernatants on Candida spp. About 60 % and 80 % of Candida isolates were recovered from saliva samples of 20 patients with fixed orthodontic appliances, and 20 children having dental caries, respectively. The antifungal susceptibility of Candida spp. was investigated using disk diffusion assay. C. albicans strains showed low resistance to fluconazole (15 %) and amphotericin B (10 %). Using the agar well diffusion assay, both L. acidophilus and L. plantarum supernatants inhibited the in vitro growth of all tested Candida spp. with inhibition zone diameters of (11-19 mm) and (7-16 mm), respectively. On observing the effects of L. plantarum and L. acidophilus supernatants on Candida cells’ morphology; the light microscopic examination demonstrated the inhibition in germ tube formation of all tested C. albicans with percentages of 68 % and 53 %, and for C. krusei with inhibition percentages of 69 % and 59 %, respectively. L. acidophilus and L. plantarum strains showed high co-aggregation ability with C. albicans strains with ranges of 42-49 % and 30-35 %, respectively. The antibiofilm activities of the two Lactobacillus supernatants were determined using the tissue culture plate assays. Signiﬁcant inhibition of bioﬁlms formation by Candida spp. was recorded on treatment with L. plantarum and L. acidophilus supernatants, with reduction percentages of 50-72 % and 74-85 %, respectively.


Introduction
Oral microbiota is defined as oral microorganisms such as bacteria, yeasts and viruses that form a contradictory ecosystem in the mouth (Lu et al., 2019). A recent study conducted by Razi et al., (2020) revealed that the oral cavity provides a nourishing environment for oral microbiota and regulates the bacterial colonization to prevent the invasion of the pathogenic microbes. The oral microbiota plays a key Novel Research in Microbiology Journal, 2021 role in maintaining the oral health. However, in some situations, the invading microbes can cause imbalances in the commensal microbial community of the mouth, leading to dental diseases (Marsh and Zaura, 2017). Caries is an infectious microbial disease of the tooth that results in the local dissolution and destruction of the calcified tissues (Heymann et al., 2013). Caries occurs due to complex interactions between dental structures and the oral microbiome; biofilm formation, food residue accumulation, saliva dysfunction and genetic predisposition (Zero, 2006).
In the oral cavity, Candida spp. represent commensal yeasts that could contribute to the formation of a complex oral microbial biofilm. The colonization rate of Candida spp. is 20-40 % in healthy individuals and 60 % in immuno-compromised persons, as they become the most prevalent microflora (Signoretto et al., 2009). Increase in the intake of sweet diet; poor oral hygiene and the presence of carious lesions in children, support oral colonization by Candida spp. (Shino et al., 2016). C. albicans is known to be associated with dental caries, but the role of non-Candida albicans including C. krusei and C. tropicalis in the development of dental caries is recently described by Beena, (2020).
Fixed orthodontic appliances are artificial devices in the mouth that greatly affect the health of the mouth and allow the accumulation of plaques and food debris. They can bring a large number of microorganisms with associated infections to the mouth (Atack et al., 1996). The previous study of Heintze et al., (1999) revealed that during the treatment of fixed orthodontic appliances, festive regions are formed that are suitable for biofilms accumulation. Badiee et al., (2011);Castanheira et al., (2014) added that increased dental plaque levels are associated with the development of gingivitis. Patients with gingivitis are liable to suffer from periodontal disease. After application of the fixed orthodontic appliances, the plaque pH and the number of microorganisms in the oral cavity change. C. albicans and other Candida strains aggregate or adhere much more easily to the fixed orthodontic appliances.
The antifungal drugs are very effective however they have many side effects especially the antifungal resistance (Sardi et al., 2013). The antimicrobial agents act non-specifically through decreasing the levels of beneficial and harmful oral microorganisms. According to Mishra et al., (2016), Chlorhexidine is a broad-spectrum powerful antimicrobial agent but it has side effects such as staining of teeth and unpleasant taste that restrict its utilization as a rinse for a longterm. Probiotics are living bacteria, derived from the genus Lactobacillus or Bifidobacterium (Doron and Gorbach, 2006). A previous study conducted by Mishra et al., (2016) highlighted that probiotics provide a natural protection against the bacteria that are harmful to the teeth and gums; by utilizing the beneficial naturally occurring oral bacteria, thereby maintaining a healthy microbial balance in the oral cavity. The adherence ability of the microorganisms belonging to the same species (auto-aggregation), and ability of the genetically different microorganisms to adhere to each other (co-aggregation); are considered as preliminary screening protocols for detecting the probiotics. Moreover, these abilities are important for the development of oral biofilms, and help in providing protection to the microbiota against shear forces, which occur naturally in the oral cavity (Ledder et al., 2008).
According to Pandya, (2016), probiotics help to prevent and treat oral disorders through different mechanisms including; inhibition of pathogen adhesions and biofilm formation, elimination of competitors; affect plaque development and its complex ecosystem by competition with the adherent bacteria, production of chemicals such as organic acids, hydrogen peroxide and bacteriocins that suppress the oral bacteria, regulation of systemic immune function, affecting the local immunity, regulation of mucosal permeability acting as antioxidants, and inhibition of plaque induction by neutralizing the free electrons.
The objectives of this study were to identify Candida spp. that accompany placement of the fixed orthodontic appliances and dental caries, and to evaluate the effectiveness of using probiotics as antifungal agents; to reduce biofilms formation by Candida spp. in the oral cavity.

Patients and samples
Patients selected for this study were 20 children with dental caries from Pedodontics Department, whereas the other 20 patients had fixed orthodontic appliances, attended for periodical provision in the Outpatient Clinics of Department of Orthodontics, Faculty of Dentistry, Minia University, Minia, Egypt. All patients or children's parents gave their written consent to participate in this study, and were informed them that the use of antimicrobial mouthwashes was banned during the study. Saliva samples were collected from children with caries and from patients using orthodontic appliances; 6 months after starting the orthodontic therapy. The patients were instructed individually to expel saliva into a sterile container until a volume of approximately 3 ml was collected.

Microorganisms and identification of Candida spp.
About 100 µl of each saliva sample was cultured on Sabouraud dextrose agar (SDA) (Lab M, UK) and CHROMagar Candida (CHROMagar Candida, France) media, and then incubated at 35˚C for 48 h. Generally, CHROM agar Candida medium identifies Candida spp. by their color and growth pattern (Nadeem et al., 2010). For phenotypic differentiation between Candida spp., the germ tube test was performed according to Benson, (2002). A small portion of the isolated colonies of the tested Candida spp. was suspended individually in 0.5 ml of human serum in a test tube, and then incubated at 35˚C for 2h. After incubation, a drop of Candida serum suspension was placed on a slide; covered with a coverslip, and then checked microscopically for the presence of germ tubes. The probiotic strains of L. plantarum ATCC 14917 and L. acidophilus ATCC 20552 were provided by the MIRCIN culture collection of the Faculty of Agriculture, Ain Shams University, Egypt.

Antifungal susceptibility assay
The in vitro activities of the different antifungal agents against the isolated Candida spp. were measured using the disk diffusion assay, as described by Ng et al., (2001). Overnight cultures of Candida spp. were adjusted to 0.5 McFarland turbidity standards, and then 0.5 ml of each culture suspension was spread individually over the surface of Muller-Hinton agar (MHA) (Lab M, India) plates supplemented with 2 % glucose, using a sterile glass spreader. The antifungal disks used were ketoconazole (10 µg), fluconazole (25 µg) and amphotericin B (20 units) (Bio-analyse R, Turkey). These disks were placed on the surface of MHA and then all plates were incubated at 37°C. The inhibition zone diameters were measured using a calibrated ruler after 24h of incubation.

Detection of antifungal potential of the Lactobacillus supernatants against Candida spp.
The antifungal potentials of L. plantarum ATCC 14917 and L. acidophilus ATCC 20552 supernatants against the recovered Candida isolates were examined using the agar well diffusion technique, in reference to Toba et al., (1991). The two Lactobacillus strains were incubated in de Man Rogosa Sharp (MRS) broth at 37°C for 48h. After incubation, the cultures were centrifuged at 10.000 rpm for 10 min.; the supernatants were filtered through a 0.2 μm filter (Aly et al., 2018). On the other hand, overnight cultures of the Candida isolates were adjusted to 0.5 McFarland turbidity standards using physiological saline. About 100 μl of each Candida suspensions was spread individually over the surface of SDA agar plates using a sterile glass spreader. Wells of 10 mm were made in these seeded SDA plates using a sterile cork borer, and then 100 μl of each Lactobacillus supernatant was added individually to each well. The plates were incubated at 35 °C for 48h. Finally, zones of inhibition formed around the wells were measured using a calibrated ruler, and then compared to MRS broth control wells. Three replicates were used for each Novel Research in Microbiology Journal, 2021 Lactobacillus supernatant and the test was repeated thrice.

Effect of Lactobacilli supernatants on Candida spp. germ-tube formation
Tested Candida spp. were adjusted to a cell density of 0.5 McFarland in human serum, and then added individually to equal volume of supernatants of the 2 Lactobacillus strains, whereas Candida cells without Lactobacillus supernatants were used as negative control. The tubes were incubated at 37°C for 2 h. After incubation, about 100 Candida cells from treated samples and negative control were observed for the presence of germ tubes using a light microscope, according to the method adopted by Liu et al., (1994). The percentage of inhibition in Candida cells' germination was calculated in three independent assays.

Surface hydrophobicity of Lactobacillus strains
The surface hydrophobicity of cells of the two Lactobacillus strains was investigated using the Salt aggregation test (SAT), as described by Andreu et al., (1995). Bacterial cells were suspended in phosphate buffered saline (PBS) (pH 6.8) to a final concentration of 5 × 10 9 cfu/ ml. The bacterial suspensions were mixed individually on a glass slide with equal volumes of different concentrations (0.5, 1.5, 2 and 4 mol/ l) of Ammonium sulfate solution. The lowest concentration of Ammonium sulfate that caused visible aggregation of the Lactobacillus cells was defined as the SAT hydrophobicity values. Based on these values, the tested bacterial strains were classified as high hydrophobic (< 0.9 mol/ l), intermediate hydrophobic (0.9-1.5 mol/ l), or hydrophilic (>1.5 mol/ l). For comparison, negative and positive controls were used by mixing equal volumes of the bacterial suspensions with 0.02 M phosphate buffer (pH 6.8) and 4 M Ammonium sulfate solution, respectively. The test was performed in triplicates and repeated twice.

Auto-aggregation assay of Lactobacillus strains and Co-aggregation of Lactobacillus strains with Candida spp.
The auto-aggregation assay was done according to the method conducted by Schillinger and Lücke, (1989). L. acidophilus, L. plantarum and Candida cultures were adjusted to 5 × 10 9 cfu/ ml concentration with PBS at pH = 6.8, and the absorbance of each suspension was measured at 600 nm (initial aggregation index). These suspensions were incubated at 37 ºC, and then assessed at intervals of 4, 20 and 24h (final aggregation). The auto-aggregation index was calculated according to Schillinger and Lücke, (1989) as follows: Aggregation % = 100 x (A initial -A final )/A initial Where; Aggregation% = Aggregation index, A initial = initial absorbance at 600 nm (at 0 h), A final = final absorbance at 600 nm (at 4 h, 20 h and 24 h according to the assay) The co-aggregation assay was carried out to detect the ability of Lactobacillus strains to co-aggregate with the tested Candida spp., in reference to Reid et al., (1990). Lactobacilli and Candida spp. were suspended individually in PBS (pH = 6.8), and then adjusted to a final concentration of 5 × 10 9 cfu/ ml. Equal volumes of Lactobacilli and Candida spp. were mixed and then incubated at 37 °C for 24 h. Each assay was performed in triplicates. About 2 ml of each Lactobacillus strain and Candida sp. were used as controls. Absorbance (A 600 nm) of the mixtures was measured at 4, 20 and 24 h, and the co-aggregation percentage was calculated according to Ekmekci et al., (2009) as: Where; A Candida , A Lactobacillus and Amix represent the absorbance at 600 nm of the control tubes of Candida spp., Lactobacillus strains and their mixture; respectively, after incubation for the pre-determined time intervals.

Detection of biofilm formation by Candida spp.
Biofilm formation by the Candida isolates was detected using the tissue culture plate method, according to Stepanovic et al., (2000). The Candida isolates were cultured on SDA at 37 °C for 24 h.
Cultures were adjusted with SDB to 10 7 cfu\ ml. An aliquot of 20 μl was distributed individually in each well of a 96-well plate. After that, about 180 μl SDB containing 2.5 % glucose was added, and then the plate was incubated for 48h at 35 °C. After incubation, the wells were washed 3 times using sterile physiological saline, and then fixed with 200 μl of methanol (99 %) for 15 min. At the end of this fixation period, excess methanol was discarded and the wells were left to dry. After dryness, the wells were stained with 200 μl of crystal violet (2 %) for 5 min., washed with dist. water and then dried. The wells were treated with 160 μl of glacial acetic acid (33 %), and then the optical density (OD 540 ) was measured spectrophotometrically. Biofilm formation was evaluated according to the measured OD, where; negative (-) if OD values were 0 ≤ OD 540 ≤ 0.120, weak (+) if 0.121≥ OD 540 ≤ 0.240, intermediate (++) if 0.241 ≤ OD 540 ≤ 0.500, and strong biofilm former (+++) if OD 540 ≥ 0.500. The assay was carried out in duplicates.

Inhibitory effects of
Lactobacillus supernatants on the strong biofilm forming Candida spp.

Effect of Lactobacillus supernatant on biofilm formation by Candida spp.
To test the effect of filtrates of the 2 Lactobacilli strains (L. plantarum ATCC 14917 and L. acidophilus ATCC 20552) on biofilm formation by the strong biofilm forming Candida isolates, an aliquot of 10 μl of Candida isolates pre-cultured in SDB (10 7 cfu/ ml) was distributed individually into the wells of 96-well microtiter plate. After that, about 140 μl of SDB containing 2.5 % glucose was added onto each well. A volume of 50 μl of sub-MIC (minimum inhibitory concentration) of the 2 Lactobacillus filtrates was added individually to each well, and then the plate was incubated at 35°C for 48 h. Candida isolates and SDB were used solely as controls. The degree of biofilm formation was assessed according to the tissue culture plate method described above.

Effect of Lactobacillus supernatants on reduction of pre-formed biofilms by Candida spp.
For testing for the inhibitory effects of Lactobacillus supernatants on the preformed Candida biofilms; Candida biofilms were formed in the 96-well microtiter plates for 24 h, subsequently 100 µl of the Lactobacilli supernatants was added individually to these preformed biofilms, and then incubated for another 24h. The percent reduction in biofilm formation was calculated according to the following equations, adopted by Kaur et al., (2018): Percentage inhibition = 100 -(OD 540 of test wells/ OD 540 of control wells) ×100

Statistical analysis
Statistical analysis of results was carried out using SPSS version 17.0 (SPSS Inc., Chicago, IL). The antibiofilm activities were analyzed through One-way analysis of variance (ANOVA) and Tukey's multiplecomparison test. Results with a p-value less than 0.05 were considered statistically significant.

Isolation and identification of Candida spp.
Out of 20 orthodontic samples, 12 (60%) Candida isolates are recovered and identified as; C. albicans (8), C. tropicalis (2) and C. krusei (2). Whereas 16 (80%) Candida isolates are recovered from 20 children having dental caries, and are identified as; C. albicans (12), C. tropicalis (3) and C. krusei (1). On CHROMagar, C. albicans are observed as green smooth colonies, C. tropicalis as blue raised colonies and C. krusei as pink fuzzy colonies. However, C. albicans is the most prevalent species recovered from both types of samples as shown in Table (1). Results of germ tube test confirmed that both of C. albicans and C. krusei isolates are germ tube formers.

Antifungal susceptibility of the Candida spp.
The antifungal susceptibility patterns of the 28 Candida spp. to ketoconazole, fluconazole and amphotericin B antibiotics are shown in Table (2). C. tropicalis showed higher resistance rate (40 %) to fluconazole, followed by C. albicans (15 %) and C. krusei (33.3 %), while C. krusei demonstrated higher resistance (33.3 %) to amphotericin B. On the other hand, C. albicans and C. tropicalis presented the highest resistance against ketoconazole (40 %).

Antifungal potential of Lactobacillus supernatants
The antifungal activity of Lactobacillus strains was tested using agar well diffusion assay. Both L. plantarum ATCC 14917 and L. acidophilus ATCC 20552 supernatants inhibited the growth of all tested Candida spp. As shown in Table (3), L. acidophilus has higher antifungal efficacy than L. plantarum against C. albicans; the inhibition zone diameter ranged from 14.2 to 19.7 mm, followed by C. tropicalis (11.9-16.6 mm) and C. krusei (11-12.1 mm).

Effect of Lactobacillus supernatants on Candida spp. germ-tube formation
By examination under the light microscope, it is observed that supernatants of both Lactobacillus strains inhibited the germination ability of all the tested C. albicans and C. krusei. As shown in Fig. (1), the percentage of germ tube formation decreased on using the L. plantarum and L. acidophilus supernatants after 2 h of incubation by 68 % and 69 % for C. albicans, compared to the control cells. On the other hand, Fig. (2) shows that the germination inhibition percentage of C. krusei cells on treatment with L. plantarum is 53 %, whereas it is 59 % in case of L. acidophilus, after 2 h of incubation.
Novel Research in Microbiology Journal, 2021

Surface hydrophobicity, auto-aggregation, and co-aggregation of Lactobacilli with Candida spp.
The SAT was used to investigate the hydrophobic/hydrophilic properties of Lactobacillus surfaces. L. plantarum ATCC 14917 and L. acidophilus ATCC 20552 showed hydrophilic properties, as cell aggregates are formed with both tested Lactobacillus strains after 1 min. at a salt concentration of 2 mol/ l. Much larger aggregates are observed with L. acidophilus than L. plantarum.
Regarding auto-aggregation test, the tested Lactobacillus strains showed high ability to autoaggregate. L. acidophilus expressed higher percentage of auto-aggregation after 24 h (71 %) than that recorded by L. plantarum (53 %). Auto-aggregation increased with increasing the incubation time, and the highest auto-aggregation is recorded at 24 h, as shown in Table (4).
Also, all the tested Candida spp. auto-aggregated and the ranges of auto-aggregation percentage are; 24 -27 %, 22-26 % and 23-29 % after 24 h, for C. albicans, C. tropicalis and C. krusei, respectively. The autoaggregation percentage of the 2 Lactobacillus strains is much higher than the Candida spp. auto-aggregation, tested under the same conditions. According to results of the Co-aggregation assay, both of L. acidophilus and L. plantarum showed high ability to co-aggregation with all the tested Candida spp. at different percentages; after 4 h, 20 h and 24 h of incubation, as demonstrated in Table 5. The highest co-aggregation percentages of L. acidophilus and L. plantarum are recorded with C. albicans with ranges of 42±5.2-49±3.2 and 30±3.7-35±0.7; respectively, as demonstrated in Table (5).   Where; (*) Represent means of Co-aggregation percentages; (±): SD; (**): Number of strains; (***): Range of co-aggregation percentages for the tested isolates

Ability of biofilm formation by Candida spp.
Biofilm formation by Candida spp. was detected using the tissue culture plate assay. Out of 28 Candida isolates, 66.7% of C. krusei showed strong biofilm formation, followed by C. albicans and C. tropicalis (both 20 %). Conversely, weak or no biofilm formation is observed in 50 %, 40 % and 33.3 % of C. albicans, C. tropicalis and C. krusei; respectively, as shown in Table (6). The strong biofilm forming Candida spp. are C. albicans (1, 2, 6, 12), C. tropicalis (2) and C. krusei (1 and 3). Antibiofilm activity of L. plantarum and L. acidophilus supernatants was tested on both of biofilm formation and mature preformed biofilm by the strong biofilm forming Candida spp. including; C. albicans (1, 2, 6, 12), C. tropicalis (2) and C. krusei (1 and 3). As demonstrated in Fig. (3), significant biofilm inhibition is recorded when Candida spp. were treated with L. plantarum and L. acidophilus supernatants, with percentage inhibition of 50-72 % and 74-85 %, respectively. On studying the effects of the two Lactobacillus supernatants on reduction of the preformed biofilms, it is observed that the supernatants effectively eradicated the preformed Candida biofilms, but at lower percentages than those recorded during the inhibition of biofilm formation. The tissue culture assay expressed a reduction percentages of 26-35 % and 29-44 % for L. plantarum and L. acidophilus supernatants; respectively, as presented in Fig. (4).

Discussion
Treatment with fixed orthodontic devices is associated with significant biofilm accumulation, thus patients are at risk of deteriorating their oral health, as confirmed by Hadj-Hamou et al., (2020). In this study, 60 % and 80 % Candida spp. were isolated from orthodontic and dental caries samples; respectively, and 3 different species were identified mainly; C. albicans (40 % and 60 %), C. tropicalis (10 % and 15 %) and C. krusei (10 % and 5 %), respectively. In consistence with the current results, Al-Oebady et al., (2019) showed higher colonization by Candida spp. in presence of fixed orthodontic devices; compared to the normal flora, and C. albicans was present at a percentage of 35 %, followed by C. tropicals (23.7 %), C. parapsilosis (21.1 %) and C. krusei (19.5 %). In a similar recent study, Alhasani et al., (2020) recorded that C. albicans (72.5 %) was the most commonly recovered yeast sp., which colonized the oral cavity after introduction of fixed orthodontic appliances, followed by C. glabrata and C. tropicalis (both 12.5 %). Moreover and consistent with our results, Mishra et al., (2016) found that the prevalence of C. albicans was 69.2 % in children with decayed teeth. In addition, Al-hebshi et al., (2015) revealed that Candida spp. were detected in 63.3 % of the children, where C. albicans represented 69 % of the total isolates, followed by C. tropicalis, C. glabrata, C. krusei and unidentified species, which recorded prevalence of 11.8 %, 5.5 %, 2.3 % and 11.4%, respectively.
Recently, Radi and Abdelmonem, (2017) proved that emergence of resistance to the antifungals is a serious health problem. In this research work, the antifungal sensitivity of Candida spp. was investigated using the disk diffusion method. We found that C. tropicalis and C. krusei showed the highest resistance to fluconazole (40 % and 33.3 %) and amphotericin B (20 % and 33.3 %); respectively, while both of C. albicans and C. tropicalis expressed the highest resistance to ketoconazole (40 %). In contrast to these results; Alhasani et al., (2020) demonstrated that all oral isolates of Candida spp. were susceptible to amphotericin B and ketoconazole; however, in accordance with the present study, resistance to fluconazole was recorded in C. tropicalis (40 %) and C. albicans (13.8 %). Compared to this study, recent study conducted by Al-Oebady et al., (2019) revealed higher rate of resistance to amphotericin B in C. krusei (60 %), while the ketoconazole resistance rates were 50 %, 75 % and 50 % for C. albicans, C. tropicalis and C. krusei, respectively.
As reported recently by Hadj-Hamou et al., (2020), the use of probiotics is proposed to prevent and/or treat oral pathologies such as dental caries and periodontal tissues diseases. In this study, the antifungal activities of Lactobacillus supernatants were assessed using agar well diffusion assay. Results showed that all tested Candida spp. were inhibited by supernatants of both probiotic bacteria, where L. acidophilus had higher antifungal efficacy than L. plantarum recording inhibition zone diameters ranging from 11.9-19.7 mm. Current results are consistent with those of the previous study of Radi and Abdelmonem, (2017), who highlighted that L. acidophilus had the most effective antifungal efficacy against both C. albicans and non-albicans (67.5 %), followed by L. rhamnosus (41 %) and L. casei (15.5 %); however, L. plantarum had the least antifungal potential (9 %). Furthermore, Salari and Almani, (2020) recently investigated the antifungal potencies of cell-free supernatants of various concentrations of L. acidophilus and L. plantarum on five oral Candida spp., and found that C. albicans was the most sensitive to the tested Lactobacilli supernatants.
We demonstrated the inhibitory effects of Lactobacillus supernatants on germ tube formation, as germination of Candida spp. is a virulent determinant. Current results proved that the two L. plantarum and L. acidophilus supernatants suppressed the formation of germ tubes by the tested C. albicans (recording inhibitions of 68 % and 53 %), and C. krusei (69 % and 59 %), respectively. In accordance, a previous study conducted by Noverr and Huffnagle, (2004) investigated the effects of supernatants obtained from 2 h probiotic bacterial cultures on the morphology of C. albicans. They observed the inhibition of C. albicans germ tube formation, while inclusion of 24h cultures completely inhibited this germination. They attributed this inhibitory activity to the presence and accumulation of soluble compounds in the culture supernatants.
In this study, the ability of biofilm formation by the oral Candida spp. was detected using the tissue culture plate assay; where C. krusei, C. albicans and C. tropicalis, exhibited strong biofilm formation (66.7 %, 20 % and 20 %, respectively). Similarly, the recent research work conducted by Alhasani et al., (2020) tested the ability of the isolated Candida spp. to form biofilm using the same assay, and recorded biofilm formation rate of 52.5 % for all the tested of Candida spp.; however, biofilm formation occurred more frequently among C. tropicalis and C. glabrata (both 60 %), than in C. albicans (48.3 %).
The current study revealed significant inhibition of biofilm formation by the strong forming Candida spp. when treated with L. plantarum supernatant, although this inhibition was lower than that recorded by L. acidophilus. Similarly, a recent study conducted by Tan et al., (2018) proved that L. acidophilus cell-free supernatant inhibited C. albicans biofilm development and filamentation.
Bacterial surface hydrophobicity is the main mechanism of adhesion in the mouth (Rosenberg et al., 1983). In the present study, cells of L. plantarum and L. acidophilus strains showed hydrophilic properties on testing with the salt aggregation test (SAT). Similarly, the hydrophilic nature of Lactobacilli had also been demonstrated in the previous studies conducted by Deepika et al. (2009);Gong et al., (2012). However, in contrast to our findings, Piwat et al., (2015) stated that most oral Lactobacilli had moderate to high hydrophobicity. This disagreement in results may be attributed to the difference in the methods used to detect the bacterial hydrophobicity, as reported by Marin et al., (1997). A previous study of Van Loosdrecht et al., (1987) have proved that for hydrophobic microorganisms, hydrophobicity is the primary factor controlling adhesion, although adhesions in case of hydrophilic microorganisms are dominated by an electrokinetic force.
Our study showed that L. acidophilus demonstrated higher percentage of auto-aggregation (71 %) compared to L. plantarum strain (53 %) after overnight incubation. The tested Lactobacillus strains showed high ability for co-aggregation with all the Candida spp. after 24 h of incubation; however, the highest co-aggregation percentages were recorded by L. acidophilus and L. plantarum with C. albicans isolates with range of percentages of 42-49 % and 30-35%, respectively.
Similar to the present results, Chervinets et al., (2018) showed that tested Lactobacillus strains made auto-aggregation and co-aggregation with Candida spp., and exhibited high surface hydrophobicity. Sazawal et al., (2006);Chervinets et al., (2018) reported that auto-aggregation, co-aggregation and surface hydrophobicity are among the most important features of the adaptive potential, which are the basis of biofilm formation. An earlier studies conducted by Al-Ahmad et al., (2007); Ledder et al., (2008) proved that aggregation aids in the entry of new bacterial species within the biofilms, thus the exchange of genes and metabolic products will be easier and supports the microorganisms' survival in different environments. Chervinets et al., (2018) recently revealed that Lactobacilli incorporated in biofilms can easily synthesize substances that resemble antibiotics, which suppress the growth and proliferation of pathogenic and opportunistic microflora; to ensure survival of Lactobacilli, protection and increase in number. Moreover, a previous study of Vilela et al., (2015) highlighted that formation of biofilms is probably reduced by probiotics through the production of inhibitory substances called "bacteriocins". Later, Wannun et al., (2016) reported the isolation of a bacteriocin called "fermencin SD11" from L. fermentum SD11, which is a human oral Lactobacillus that has a powerful inhibitory effect on oral Candida cells. The anti-Candida activities can be attributed to several reasons including; co-aggregation, H 2 O 2 production and oral pH modification (Jørgensen et al., 2017), through releasing large amounts of lactic acid (Denkova et al., 2013), and through complete inhibition of fungal biofilms formation (Chew et al., 2015).

Conclusion
We concluded that orthodontic treatment with fixed appliances and dental caries caused specific alterations in the oral environment. Candida spp. are opportunistic microorganisms that showed resistance to several antifungal drugs and can form biofilms in the oral cavity. Results of this study indicated that attention has to be paid to control the oral Candida infection. Probiotic Lactobacilli have good adaptive properties; antimicrobial and antibiofilm potentials against oral Candida. Thus they can be used in prevention and control of these yeast infections. Further investigations should be carried out to assess the characteristics of the probiotic strains before providing them for clinical trials. Screening and characterization of Lactobacillus strains are necessary to discover ideal and novel probiotics.