Plant material
The plant material used in this study consisted of three plants including Acalypha wilkesiana (AA6752HNB), Senna alata (YH410HNB) and Mitracarpus hirtus (YH388HNB) collected in the commune of Abomey-Calavi (Benin) in their natural habitat and authenticated at the National Herbarium of Benin.
Biological material
The biological material consisted of eight Candida isolates collected from stool of people living with HIV (PLWHIV) and a reference strain of Candida albicans ATCC 90028 (Ref. 0264P Microbiologics, Paris, France).
Phenotipic identification of Candida strains
Phenotypic identification of the strains was performed using the technique of Ghaddar et al. [7].
Identification of Candida strains on chromogenic media ChromAgar and Tetrazolium Reduction Medium
The method of Giri and Kindo. [8] was used. Indeed, the coupled aspects of two chromogenic media were concomitantly noted. These chromogenic media allow the identification of species of the genus Candida from the different colors, morphologies, textures presented by the colonies. Thus, a fraction of isolated colonies of each strain was plated on Chromagar Candida (Conda, Spain) and Tetrazolium Reduction Medium (TRM), then the cultures were incubated at 37°C for 48h. Aspects of the derived colonies were recorded, and the strains identified. The following correspondence table (Table I) was used for strain classification.
Table I: Identification aspects of Candida strains on ChromAgar (Conda, Spain) and on TRM medium
Candida strains
|
Colony appearance
|
ChromAgar
|
TRM
|
Candida albicans
|
Petites colonies vert clair
|
Small to medium pinkish colonies
|
Candida glabrata
|
Small pinkish-gray colonies rosâtre
|
Small shiny pink colonies
|
Candida
Parparapsilosis
|
Pink-orange colonies
|
Pink-orange colonies
|
Candida krusei
|
Large pale pink colonies
|
whitish outline Small to medium pinkish and dry colonies
|
Candida tropicalis
|
Blue or metallic blue
|
violet colonies Dry brown to violet colonies
|
Candida spp.
|
small to medium pink colonies
|
purple colonies
|
Blast test (filamentation test)
Each isolate was cultured in blood serum (filamentation test) and incubated at 37°C. This simple test is used to study the capacity of yeasts to produce germ tubes, which is considered a virulence trait. Indeed, it is performed by emulsifying a portion of the yeast colony to be studied in 0.5ml of human blood serum contained in a test tube and incubated for 3h [9]. Then, a drop of the suspension is observed between slide and coverslip under a light microscope at X40 objective after the incubation time. A positive result is the appearance of filament starting from a cell without constriction. It allows to differentiate Candida albicans from non-Candida albicans.
Research of virulence factors
Several types of virulence factors were investigated including phospholipase production, gelatinase production, hemolysin production, biofilm production or RCA test, adhesion power and hydrophobicity power. For these tests, a suspension was prepared by taking an isolated fresh colony obtained on SDA + chloramphenicol agar in 2ml of sterile YEPD broth and incubated at 30°C. After 18h incubation, the culture was centrifuged and washed twice with sterile PBS at 3000g for 5 minutes. After removing the supernatant in the last wash, 1ml of PBS was added to the pellet and resuspended by vortex. The resulting fungal suspension was diluted to obtain a solution with an optical density between 0.4 and 0.5 corresponding to a cell concentration of about 107 CFU/ml.
Production of phospholipases or lecithinase
The fungal suspension made at 107 CFU is inoculated on a petri dish containing SDA, yeast extract, NaCl, CaCl2 added with 10% egg yolk. The dish is placed at 37°C in the oven for 120h to 7 days. An opaque zone around the grown colony leads to a positive result. The ratio of the colony diameter to the opaque zone is compared with the reading grid described by Sriphannam et al. [10].
- Pz < 0.40 corresponds to high production,
- Pz between 0.41-0.60 corresponds to moderate production,
- Pz between 0.61-0.80 corresponds to low production,
- Pz between 0.81-0.99 corresponds to a very low production,
- Pz =1 corresponds to no production,
Gelatinase production
The gelatinase production assay was put according to Elavarashi et al. [11]. An 18h young colony isolated on YEPD (Yeast Extract Peptone Dextrose) agar was taken, seeded into 4 ml of sterile nutrient gelatin agar (5 ml special peptone 3g/l beef extract and 120g/l gelatin) in tube and incubated at room temperature (25 oC) for 2 weeks and examined at the regular time interval to check the liquefaction of gelatin. A strain of Staphylococcus aureus was used as a positive control, the negative control is made up of tubes containing only the agar medium. Gelatin liquefies at over 28 o C generally, to confirm gelatin liquefaction due to gelatin production, test and control tubes were placed at 4 oC in the refrigerator for 30 minutes. The tubes were tilted to observe the liquefaction of the gelatin. The hydrolyzed gelatin remains liquid even after gelatin production is demonstrated by cold exposure while the uninoculated tubes remained in the solid state. Thus we have gelatinase positive : liquefied medium and gelatinase negative : solid medium.
Hemolysin research on blood agar
This research of hemolysin production by the strains is done on fresh blood agar and incubated at 37o C for 48h. The test is positive if there is a clear zone around the colony.
Assessment of adhesion strength and biofilm formation
At this level, a cell suspension is prepared according to the modified method of Noumi et al. [12]. Indeed, it was performed a dilution of the fungal cell concentrate in PBS (centrifugation pellet + 500µL of PBS) and in RPMI 1640 medium to have the density of the suspension obtained adjusted to 0.38 at 520 nm corresponding to 107UFC/ml (50µL of the PBS suspension). This cell concentration is the one required for the evaluation of adhesion and biofilm formation power according to Nakamura-Vasconcelos et al. [13]. Four plates (96 wells) with flat bottom were used two by two for adhesion power and biofilm formation respectively with RPMI 1640 medium and YEPD medium. For each type of medium, the attached plating plan was followed. Introduction into wells A1 and A2 (negative control). Introduction of 200µL of sterile RPMI in wells A3 and A4 (or sterile YEPD) as a second negative control. Three consecutive wells are used for one strain. Thus, the first sample occupied wells A5, A6 and A7. In the first well A5, it was transferred 200µL of the suspension made with PBS. As for the next two wells A6 and A7, 200µL of the suspension made with RPMI 1640 medium (or YEPD) was transferred. The treated plates were incubated for 2 hours at 37°C on a rotary shaker at a speed of 75 rpm. After the incubation time, the RPMI 1640 medium was aspirated, and the non-adherent cells were washed twice with PBS.
Adhesion power
It was transferred in all the wells of one of the plates initially prepare 100µL of a 1% Crystal violet solution to stain them. After the minute of action of the dye, the wells were immediately washed with PBS and dried in the oven for 1h. The evaluation of the adhesion is done either directly by comparing the stains of the wells or indirectly by reading the OD (570 nm) of the supernatant of the well wash solution to which 200µL of glacial acetic acid or alcohol-acetone has been added. Thus, the following classifications were considered by comparing the optical densities read according to Noumi et al. [12]. The power of adhesion was determined according to the following criteria. Non-adherent (0) = OD ≤ Doc ; Low adhesion (+) = ODc < OD ≤ 2 x ODc ; Medium adhesion (++) = 2 x ODc < OD ≤ 4 x ODc, High adherence (+++) = 4 x DOc < DO.
Biofilm formation
Quantitative assessment of biofilm formation was done according to the modified protocol of Noumi et al. [12]. Considering here the second plate, a total of 100µL of YNB medium was transferred to each washed well using a pipette. The plate was incubated at 37°C on a shaker at 75rpm. During the 66 hours that biofilms were growing, the used medium was renewed daily by suctioning it with a new medium. The control wells contained only YNB medium. The wells were washed twice with PBS. Biofilms formed were quantified by colorimetry. Indeed, 100µL of a 1% Crystal violet solution was added to all wells to stain any biofilms formed. After one minute the dye was completely removed and 200µL of alcohol-acetone mixture was added to decolorize. The biofilms formed were quantified directly by comparing the shades of the wells and indirectly by reading the OD of the supernatant of the well wash solution at 570 nm with a spectrophotometer. All tests were repeated three different times. As in the case of adhesion power, the yeasts tested were classified on the basis of the same classification scale.
Assessment of cell surface hydrophobicity (CSH)
The hydrophobicity of Candida strains was measured using the protocol described by several authors ([14] ; [12]). It consists in measuring the adhesion of yeast to hydrocarbons, such as cyclohexane. Thus, as in the protocol for biofilm formation, suspensions of the different strains to be tested were prepared with phosphate buffered saline (PBS) and concentrated to obtain a solution with a density corresponding to OD620 varies between 0.4 and 0.5 (A0). For host cell surface adhesion assays, 100µL of each fungal suspension (obtained by diluting in 500µL the centrifugation pellet) was transferred to the wells of a sterile microplate (96 wells). Then, 300µL of cyclohexane was added to the wells except for the first three wells which served as negative control wells. After this treatment, the microplate was shaken for 3 minutes and left to rest for 15 to 20 minutes at room temperature. The aqueous phase is finally transferred to a new microplate and the ODs read at 620nm (A1) using a plate reader. The absorbances (A1) correspond to the measured OD of the test wells and are compared to those obtained for the control wells (negative control) OD(A0). The percentage of cells in the cyclohexane layer (stuck cells) denoted by %CSH was used to estimate the amount of absorption of the cyclohexane layer at 620 nm (A1). It was compared to that obtained before the mixing procedure (A0) of hydrophobicity using the serial dilutions of yeast mixtures will be set up on CHROMagar Candida to differentiate and recognize them. The presence of budding yeast cells with pseudohyphae under direct microscopy and yeast growth were considered as Candida strains.
Susceptibility of strains to modern antifungal agents
The sensitivity of isolates to antifungal drugs was determined by the agar diffusion method [15] (. A suspension with a density of about 0.5 Mc Farland was prepared from young colonies of the isolates to be tested. The suspension thus prepared was plated on Mueller Hinton khanagar supplemented with 2% glucose and 0.5% methylene blue (MHGB) by flooding. The excess suspension was carefully removed and the surface of the agar plates was dried before the antifungal impregnated discs were deposited. The treated MHGB agar plates were then incubated at 37°C for 24 h and the inhibition diameters were measured with a flat ruler.
Interpretations of the inhibition diameter measurements were made according to the grid in the table II [15].
Table II: Interpretation of inhibition diameter measurements
Antifungals
|
Disc concentration (mg)
|
Activity zone (mm)
|
Sensitive
|
Intermediate/
SDS
|
Resistant
|
|
|
|
|
|
Nystatine
|
100
|
≥15
|
10-14
|
≤10
|
|
|
|
|
|
Fluconazole
|
100
|
≥ 20
|
10-19
|
≤ 10
|
|
|
|
|
|
Amphotéricine B
|
50
|
>10
|
=10
|
< 10
|
|
|
|
|
|
Clotrimazole
|
10
|
≥ 20
|
10-19
|
≤ 10
|
|
|
|
|
|
Ketoconazole
|
10
|
≥ 20
|
10-19
|
≤ 10
|
|
|
|
|
|
Itraconazole
|
10
|
≥ 20
|
10-19
|
≤ 10
|
Sensitivity of Candida strains to plant extracts
Mueller Hinton agar supplemented with 2% glucose and 0.5% Methylene Blue (MHGB) and Yeast Extract Peptone Dextrose (YPD or YEPD) broth were prepared and sterilized. Three young 24-hour colonies of the test strain, grown on SDA + Chloramphenicol agar was picked and emulsified in 5mL of 9‰ saline. The resulting Candida suspensions were adjusted to density 0.5 on the Mc Farland scale corresponding to 1.5 x 105 CFU/mL. 200 mg of each extract was weighed and dissolved completely in 1mL of sterile distilled water. The resulting suspension was then thoroughly vortexed, sterilized by filtration using a syringe to which a millipore filter gauge (0.20μm and 0.22μm) is fitted, and stored in a sterile 50mL dark bottle in the refrigerator before each use. The most active extracts on inhibition of fungal strains were determined in agar medium. Three Mueller-Hinton agar plates supplemented with 2% (2 g per 100 ml) Glucose and 0.5% Methylene Blue (MHGB) were plated with the Candida suspension for each type of extract. Five 7 mm diameter wells were then sterilely made in the agar media using Pasteur pipettes. The three peripheral wells (two for the extracts and one for the positive control) were used as test wells and the central well used for the negative control. Thus, 100µL of each extract dilution (aqueous and hydroethanol extracts) of Acalypha wilkesiana Müll. & Arg., Senna alata L. and Mitracarpus hirtus (L.) DC prepared at 200 mg/mL were respectively transferred sterilely into two consecutive wells. 100 µL of Fluconazole (100µg/mL) was transferred to the last peripheral well and 100 µL of distilled water to the center well (negative control well). The treated plates were covered, left for two hours on the bench (to facilitate the diffusion of the different extracts) and then incubated at 37°C for 24 hours. The inhibition diameters observed around the wells after incubation were then measured for each type of extract studied. The most active extract corresponds to the one with the largest inhibition diameter.
Determination of the Minimum Inhibitory Concentration (MIC)
The antifungal activity of the effective extracts was determined following the methodology of Wayne, [17]. A stock solution of these extracts was prepared at the concentration of 100mg/ml in distilled water. Fluconazole at 100mg/ml was used as an antifungal agent for the positive control. Indeed, 10 µl of the fungal suspension to be tested was added to the first wells of each line of the microplate. The first two wells of each line of the microplate served respectively as negative (only the fungal suspension) and positive control (fungal suspension mixed with 100 µl of fluconazole solution). Then, 100 µl of the stock extract solution was placed in the 3rd well. Five serial dilutions of reason two were then performed starting from the 3rd well up to well 8 where the additional 100 µl was discarded. For each extract the same procedure was followed for the six different Candida strains. The microplates were covered and incubated at 37°C for 24 hours. The MICs correspond to the minimum concentrations where 50% of the yeasts are inhibited. They were estimated by counting the number of yeast using the Malassez cell under the microscope.
Data processing and statistical analysis
The data were entered using Microsoft Excel 2010. Graph Pad Prism version 8.0 was used to perform the graphs and statistical tests. Each mean was presented with standard variations for each fungal strain. With ANOVA, the means of the inhibition diameters of each extract on the fungal strains were compared with each other by Dunnett's ' multiparametric test (ANOVA). The significance level was set at 5%.