Effect of Some Non steroidal Anti-Inflammatory Drugs on Growth, Adherence and Mature Biofilms of <i>Candida spp</i><i>.</i>

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2.3. Detection of Biofilm Formation by Tissue Culture Plate Method (TCP)

The TCP assay is most widely used and was considered as standard test for detection of biofilm formation. All isolates were screened for their ability to form biofilm by the TCP method as described by Christensen et al. ^[25] with a modification in duration of incubation which was extended to 24 hours, according to O'Toole and Kolter ^[26].

2.4. Evaluation of Antifungal Activity of the tested NSAIDs Using well Diffusion Technique

Candida spp. (0.5 ml) of 1×10⁸ CFU/ml (0.5 Mcfarland turbidity) were plated in sterile petri dishes then 20 ml of sterile, molten and cooled (45°C) Muller Hinton agar media with the addition of methylene blue-glucose (to enhance zone diameter visualization) was added to all petri dishes. The plates then were rotated slowly to ensure uniform distribution of the microorganisms and then allowed to solidify on a flat surface. After solidification, four equidistant and circular wells of 10 mm diameter were carefully punched using a sterile cork bore. Each sample was applied as triplicate. The plates were allowed to stand for one hour for prediffusion of the extract to occur then incubated overnight at 37°C. All plates were examined and zones of inhibition were recorded ^{[27, 28, 29]}.

2.5. Determination of Minimum inhibitory Concentrations (MIC) of the Tested NSAIDs:

Two fold serial dilutions were performed on the tested NSAIDs. Equal volumes of each dilution were applied separately to each well in three replicates using a micropipette. All plates were incubated at 37°C for 24 hours, then the formed zones of inhibition were measured. The average of the zones of inhibition was calculated. The minimum inhibitory concentration (MIC) was calculated by plotting the natural logarithm of the concentration of each dilution of the tested NSAIDs against the square of zones of inhibition. A regression line was drawn through the points. The antilogarithm of the intercept on the logarithm of concentration axis gave the MIC value ^[29].

2.6. Germ Tube Formation

C. albicans strains were cultivated on Sabouraud’s dextrose agar for one day. Cells were suspended in sterile saline and adjusted to density of 0.5 Mcfarland turbidity. 400 µl of human serum was added to 1 ml of cell suspensions of C. albicans. Drugs were added to the suspensions to a final concentration ranged from 100 to 1000 µg/ml and saline was added to the control tube. The cell suspensions were incubated with gentle shaking at 37°C for 2 hours and were examined for the presence of germ tubes by using a light microscope. Photomicrographs of colonies and invasive growth were taken with a DMRXA microscope (Leica, Germany) ^[30].

2.7. Effect of Ketoprofen, Diclofenac Sodium, Ibuprofen on the Adherence of the Tested Candida spp. on Plastic Surfaces

All strains were first streaked onto YEPD agar (1% yeast extract, 2% Bacto peptone, 2% D-glucose, 1.5% agar) then, incubated at 25°C for 48 hours. A large loop of actively growing cells (for each strain) was transferred to sterile trypticase soy broth (TSB) (Difco Laboratories) containing 0.9% D-glucose. After incubation at 25°C for 24 hours, the cells were centrifuged and washed twice with 0.5 ml PBS (phosphate buffered saline), followed by vortexing and centrifugation at 5000 g for 5 min. The washed cells were suspended in 1 ml TSB broth and adjusted to a final OD600 nm value of 1.0 with TSB broth. These cell suspensions were then used to grow biofilms.

100 µl of the suspension (OD600) was inoculated into individual wells of polystyrene 96-well plates (flat bottom; Nunc). TSB broth was used as a negative control. The plates were incubated at 25°C for 90 min (adhesion period). Supernatants including planktonic cells were discarded and wells were gently washed with PBS twice to remove any non-adherent cells. 100 µl of fresh TSB broth containing one of, MIC, above MIC and under the MIC concentrations of each of the following solutions: Ketoprofen, ibuprofen, and Sodium Diclofenac was added to each well. The plates were covered to prevent evaporation and incubated at 25°C for 24 hours. Liquid media containing the non adherent cells were discarded through two rounds of washing with 200 µl sterile PBS buffer. Adherent cells to the plastic surfaces were quantified using Crystal violet assay. Experiment was performed in triplicate.

2.8. Effect of Ketoprofen, Diclofenac Sodium, Ibuprofen on the Preformed Biofilms of the Tested Candida spp. on Plastic Surfaces

100 µl of the suspension (OD600) was inoculated into individual wells of polystyrene 96-well plates (flat bottom; Nunc). The plates were incubated at 25°C for 48 hours. After the incubation period, the supernatants from each well were aspirated and the wells washed twice with PBS without disturbing the biofilms at the bottom of the wells, then one of sub MIC, MIC, above MIC of each of the following solutions: Ketoprofen, ibuprofen, and Sodium Diclofenac was added to wells each alone. Normal saline without any agents was added to the control wells. The plates were incubated at 25°C for 24 hours. Supernatants were discarded through two rounds of washing with 200 µl sterile PBS buffer. Cells adherent to the plastic surfaces were quantified using Crystal violet assay ^[31]. Experiment was performed in triplicate.

2.9. Scanning Electron Microscopy (SEM)

The untreated Candida spp (control) and Candida spp. treated with the tested drugs were fixed in 2.5% (vol/vol) glutaraldehyde in Dulbecco PBS (PH 7.2) for 1.5 hours, rinsed with PBS, and then dehydrated through an ethanol series. Samples were dried and gold-palladium coated. SEM examinations were made on a JSM-840 SEM (JEOL Ltd., Tokyo, Japan) ^[32].

2.10. Statistical Analysis

One-Way ANOVA as employed to evaluate any significant difference between the values obtained without the drug (controls) and the values obtained in the presence of different drug concentrations. Differences were done using SPSS version19 (SPSS Inc., Chicago, IL) for windows was used. Descriptive statistics were calculated. Kruskal-Wallis test was used to compare groups. A significant P-value was considered when it was less than 0.05.

3. Results

3.1. Biofilm Production

Four Candida spp. were tested for their ability to form biofilm and according to TCP method strains can be classified into weak adherent at OD <0.120, moderate 0.120-0.240 and strong >0.240 (Table 2)

Table 2. Biofilm production by Candida spp.

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3.2. Minimum Inhibitory Concentration of NSAIDs

Sodium Diclofenac showed a higher activity and lower MIC against C. albicans and C. glabrata while Ibuprofen showed the higher activity against C. krusei. (Table 3).

Table 3. Minimum inhibitory concentrations of Ketoprofen, Ibuprofen and Sodium Diclofenac against the tested Candida spp.

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3.3 Effect of the Tested NSAIDs on Candida spp. Membranes

Figure 1 showed the effect of Sodium Diclofenac on Candida spp. membranes. At low concentration, Pores were formed on Candida membranes (Figure 1a). At higher concentration, complete lysis and a release to the contents of cells was observed (Figure 1b).

3.4. Effect of NSAIDs on the Dimorphic Transition of C. albicans

By testing the effect of Diclofenac sodium, Ibuprofen and ketoprofen on yeast to hyphae transition of C. albicans, it was found that Diclofenac at 500 µg/ml inhibited germ tube formation while Ibuprofen and ketoprofen inhibited their formation to lesser extent in all tested concentrations (Figure 2).

3.5. Effect of Diclofenac Sodium, Ibuprofen and Ketoprofen on the Adherence And Mature Biofilms

The effect of the tested NSAIDs on adherence and mature biofilms was found to be concentration dependent (P<0.05). At MIC, Sodium Diclofenac showed the highest inhibitory activity against the adherence of C. albicans and C. glabrata but Ibuprofen showed the highest inhibitory effect on the adherence of C. krusei. On mature biofilms, Sodium Diclofenac showed the highest activity against the tested Candida spp (Figure 3, Figure 4). At 2X MIC the same results were obtained (Table 4, Table 5).

Figure 1a. At low concentration, pores were formed on the membranes of Candida cells

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Figure 1b. complete lysis to cells were observed at high concentrations

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Figure 2. Effect of the tested NSAIDs on germ tube formation by C. albicans

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Figure 3. Effect of minimuminhibitory concentration of ketoprofen, lbupofen and diclofenac sod on the adherence of Candida spp. to the tissue culture plate

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Figure 4. Effect of minimuminhibitory concentration of ketoprofen, lbupofen and diclofenac sod on mature biofilms formed by the tested Candida spp.

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Table 4. Effect of MIC, Sub-MIC and above MIC concentrations on the adherence of the tested Candida spp.

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Table 5. Effect of MIC, Sub-MIC and above MIC concentrations on mature biofilms formed by the tested Candida spp.

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