DNA extraction was performed using 24 hours young colonies isolated on YEPD medium. The chloroform-phenol technique described by Silva et al. (27) has been used. The PCR was done in two steps: the first step consists in amplifying the ITS1 and ITS4 region named ITS1-ITS4 and the second was digesting the PCR product of the ITS region with restriction enzyme. For the first step, total DNA amplification was performed using the primer pair ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’). The reaction mixture, with a total volume of 25µL, consisted of 10µL of master mix (Invitrogen), 12µL of water without DNA, 1µL of each primer and 1µL of the DNA of the yeast to be identified. The PCR was performed in a thermal cycler (PeQlab, Germany) with the following amplification conditions: 1 cycle of DNA denaturation of 5 min at 95°C, followed by 35 hybridization cycles of 30s at 94°C, 1 min at 56°C and 1 min at 72°C and a final extension step at 72°C for 10 minutes. A positive and negative control consisting of respectively a reference strain of Candida albicans ATCC 90028 and DNase, RNase free water were used.

Each PCR product was then individually digested in an aliquot with the MspI enzyme (R0106S, England Biolabs). Indeed, 10U of Msp1 enzyme corresponding to approximately 1µL of enzyme was added to 20 μl of the PCR product contained in an aliquot with 9µL of a buffer solution making a final reaction volume of 30 μl. The mixture was incubated at 37°C for 30mn and then the PCR products (ITS) and digestion products were then migrated onto agarose gels prepared at 1.5% in 0.5x TBE buffer stained with ethidium bromide, through an electrophoresis chain for 30 minutes at 100V. The observed band patterns were then observed and the observed amplicon sizes for each strain were noted and used to differentiate Candida species (28). The ITS1-ITS4 region size were presented in Table 1 .

Method described by Zanni et al. (31) which consists of measuring the adhesion of yeast to hydrocarbons such as cyclohexane, was used to evaluate the hydrophobicity of the yeast cell surface (CSH). The Optical Densities of the fungal suspensions were read and adjusted between 0.4 and 0.5 (A0). Then, 200µl of each fungal suspension to be tested was transferred into two wells of a sterile microplate to which 60µl of cyclohexane (BDH Laboratries LTD, England) was added. In the two negative control wells, the Candida suspension was replaced by distilled water. The plate was shaken vigorously for 3 min and then allowed to stand for 20 min at room temperature in a hood. Finally, 100µL of the aqueous phases from the reaction between the fungal suspensions and cyclohexane were transferred to a new microplate and the optical densities (A1) read at 620nm with the plate reader. The percentage of cells adhering to the cyclohexane layer (%CSH) was determined by the following formula:

The percentage of CSH was used to assess the importance of cell surface hydrophobicity following the criteria specified by El-Houssaini et al. (32)

The power of adhesion to polystyrene and the ability of isolates to form a biofilm on it were evaluated in this study according to the method of Zanni et al. (31) modified. Thus, we had two sterile microplates (96 wells) (one for adhesion and the other for biofilm). One hundred microliquids of RPMI 1640 medium were transferred to the test and negative control wells of a sterile microplate. 100µL of fungal suspension and 100µL of PBS solution, were added to the test and two negative control wells respectively. The treated plates were incubated at 37°C for 2 hours.

The adhesion plate was removed and the wells were washed twice with PBS solution and then stained for 1 min with 100µL of 1% diluted crystal violet. The stained wells were then washed twice with PBS solution. The treated microplate was left at room temperature for 20 min and the wells were destained for 30 min with 100µL of alcohol-acetone mixture. The supernatants were transferred to a new microplate and their optical densities were read at 560nm with a microplate reader (Infinite F200 Pro, Tecan 504).

For the biofilm plate (the second microplate), the wells were washed every 24h of incubation with sterile PBS solution and RPMI medium renewed at each occasion. The incubation time was 66h. The last wash before staining was performed after the incubation time. The adhesion and biofilm formation powers of the strains were determined from the optical densities read and then interpreted according to the criteria specified by Noumi et al. (30).

The production of phospholipase or lecithinase and gelatinase was investigated according to the methodology of El-Houssaini et al. (32) and that of Elavarashi et al. (33). 10µL of the prepared fungal suspension was seeded onto the agar and incubated at 37°C in the oven for 5-7 days. The presence of an opaque zone around the fungal colony indicates the production of lecithinase. The altering power of this enzyme (Pz) was evaluated according to the formula: Pz =D0/Dt Where, D0 = colony diameter and Dt = colony diameter + halo (large diameter). The different interpretations were made.

Gelatinase production was demonstrated according to the method described by Elavarashi et al. (33). 4000µL of gelatin-enriched nutrient broth in a hemolysis tube, were seeded with a young colony of the strain to be studied previously isolated on YEPD medium. The inoculated tubes were incubated at room temperature (25°C) and the reactions were read every 24 hours for two weeks after 30 minutes of refrigeration at 4°C. A strain of Staphylococcus aureus was used as a positive control. A tube containing only the unseeded nutrient broth was used as a negative control.

The investigation of hemolysin production by the strains was done by culturing them on 7% fresh blood (GSL) enriched agar medium (24, 32). Indeed, 10µL of the prepared fungal suspension was seeded on GSL medium and then incubated at 37°C for 48h in CO2 enriched atmosphere (5%). The presence of a greenish halo or white halo around the fungal colony shows the presence of hemolysin (alpha or beta respectively).

The medicinal plants used in this study were selected from an ethno pharmacological survey conducted in Benin on medicinal plants used in the traditional treatment of mycoses (22). These plants have been identified in the national herbarium of Benin at the University of Abomey-Calavi. These plants were collected in Houèto in the commune of Abomey-Calavi. Table 2 presents the plant material and the parts used.Three types of extracts (aqueous, hydroethanolic and ethanolic) were obtained following the protocol described by Klotoé et al. (34).

A qualitative screening of the three studied plants was carried out in order to assess the main chemical groups present according to the precipitation and colouring reactions described by Houghton and Raman (35). The main groups investigated were polyphenolic compounds (flavonoids, total phenols, tannins), anthocyanins, saponosides, reducing compounds, mucilages, and alkaloids. In the extracts produced, total polyphenols were quantified using the commercial Folin Ciocalteu reagent. Total polyphenol levels were determined using the equation from the calibration curve of gallic acid (0-200 µg/mL) taken as reference standard. Samples were prepared in triplicate for each analysis. Total polyphenol content was determined in mg gallic acid equivalent/g extract (mg GAE/g) by the formula used by Ahmed et al. (36):

With TPT, the Total Polyphenol Content, X the Gallic Acid concentration in mg/mL; V the volume of extract used in mL and m the mass of the extract in grams.

The antioxidant activity of the extracts was evaluated by the DPPH free radical scavenging assay which is one of the most widely used methods for the evaluation of antioxidant activities of herbal extracts. DPPH (2,2- Diphenyl-1-picrylhydrazyl) is a stable purplish colored free radical that absorbs at 517 nm. In the presence of anti-free radical compounds, the DPPH radical is reduced and changes its color to yellow. The method adopted in this study is that of Klotoé et al. (34). Thus 100 μL of different concentrations of each extract is added to 1900 µL of the ethanolic solution of DPPH (0.4 mg/mL). The blank is prepared by mixing 100 μL of the extraction solvent with 1900 µL of the DPPH solution. After incubation in the dark for 1 hour at room temperature, absorbance readings were taken at 517 nm using a MINDRAY spectrophotometer (BA-88-A). The recorded optical densities were used to calculate the percentage of DPPH radical scavenging which is proportional to the antioxidant power of the sample. Vitamin C and BHT were used as reference standards. Samples were prepared in triplicate for each analysis. The percentage of DPPH radical scavenging was determined by the formula:

P: Percentage of trapping; Ab: Absorbance of blank; Ae: Absorbance of sample

For the sensitivity test, seven Candida strains isolate for urines were used. These were six clinical strains of Candida and a reference strain of Candida albicans ATCC 90028. A stock concentration of 100 mg/ml of each of the nine extracts were prepared and tested separately on the in vitro growth of a reference strain of Candida albicans ATCC 90028 by the diffusion method on Muëller-Hinton agar supplemented with 2% glucose and 0.5% Methylene Blue (MHGB) (37). The fungal suspensions were prepared with Yeast Extract Peptone Dextrose (YPD or YEPD) broth. After inoculating the agar with the fungal suspension, 100 µl of each type of extract from the three plants was sterilely transferred onto Whatman paper cut into small pieces and previously deposited on the agar surface. The same procedure was followed for distilled water and fluconazole (100µg/mL) which served as negative and positive controls respectively. After 24 hours of incubation, the diameters of inhibition observed around Whatman paper were measured and interpreted. The most active plant is the one whose extracts were active on the strain with the largest inhibition diameters. The most active plant extracts were thus identified and tested following the same process on previously characterized clinical Candida strains. The anti-candida activity of the extracts was tested on the majority resistance profiles of the three most isolated Candida species ( Table 4 ).

The Minimum Inhibitory Concentrations (MIC50) of the effective extracts were determined following the methodology of Okou et al. (37) in 96-wells microplates. A stock solution of these extracts was prepared at a concentration of 100 mg/ml in distilled water. Fluconazole at 100mg/ml was used as the reference antifungal agent. 100µl of a fungal suspension made with Yeast Extract Peptone Dextrose (YEPD) (38) broth was placed in the first eight wells. The first two wells of the microplate served as positive control (YEPD broth with yeast) and reference (YEPD broth with yeast mixed with 100µl of fluconazole solution), respectively. The ninth well is the negative control containing only YEPD broth without yeast. Then, 100 µl of the stock extract solution was added to the third well. Five serial two-for-two dilutions were then performed starting from the third well to well 8 where the additional 100 µl was discarded. For each extract the same procedure was followed for six different Candida strains. The microplates were covered with aluminium foil and placed for 24 hours in an oven at 37°C. The MIC50 corresponds to the minimum concentration where 50% of the yeasts are inhibited (39). They were estimated by counting the number of yeast using the Malassez cell under a microscope with an with the X40 lens.