Collection and preservation of clinical isolates

A total of 34 Candida isolates were collected from the urine of patients admitted to Alexandria Main University Hospital, between October and December 2021. These isolates were stored in yeast extract-peptone-dextrose (YPD) broth containing 20% glycerol at -20 °C. Before use, a fresh culture was obtained by streaking an inoculum from the stock on Sabouraud Dextrose Agar (SDA, Millipore, Darmstadt, Germany), followed by incubation at 37 °C for 24 h to obtain separate pure colonies.

Antimicrobial agents and chemicals

Fluconazole (Diflucan®, 2 mg/mL IV infusion), AK (Advomikacin®, 500 mg/2 mL IV/IM vial), DEX (Dexamethasone®, 8 mg/2 mL vial), and KT (Ketolac®, 30 mg/2 mL vial) were purchased from pharmaceutical markets. Colistin sulfate and SMX powders were obtained from Pharmacure Pharmaceutical Industries and Pharco Pharmaceuticals, Alexandria, Egypt, respectively. All stock solutions were prepared by dissolving the agent in sterile distilled water (DW). For the preparation of SMX stock solution, 1 N NaOH was added dropwise until the complete dissolution of the agent.

Identification of Candida spp.Germ tube formation test

Two to three colonies of each isolate were inoculated into tryptone soy broth (Himedia, Mumbai, India), incubated at 37 °C for 2 h, then examined using a magnification power of 100X on a microscopic slide with a cover slip for the detection of germ tubes. The elongated daughter cells emerging from the round mother cells without constriction at the origin were identified as germ tubes, while those with constriction at the origin were referred to as pseudo-hyphae. Germ tube positivity, characteristic for C. albicans and C. dubliniensis, was confirmed by the presence of at least five germ tubes in the entire mount. A negative result was indicated by the absence of germ tubes in a minimum of 10 fields [63]. Positive controls, C. albicans ATCC 231GI and ATCC 10231 were included in the experiment.

Tween 80 opacity test

The Tween 80 opacity test medium was prepared by the addition of 10 g bacteriological peptone (Lab M, Lancashire, UK), 5 g NaCl, 0.1 g CaCl2, and 15 g agar to 1 L of DW. After autoclaving, the medium was cooled to about 50 °C, and then 5 mL of autoclaved Tween 80 (Alpha Chemika, Mumbai, India) were incorporated. A few overnight colonies of each isolate were used to create a circular inoculation site of a 10 mm diameter on the agar plates. The plates were incubated at 30 °C and examined daily for 10 days under transmitted light for the presence of a halo zone around the inoculum, which was recorded as a positive result indicating the ability of the isolate to produce an esterase [64]. Inoculations were performed in duplicate.

Identification with Vitek® 2 Advanced Expert System™

The identity of all isolates to the species level was confirmed using Vitek® 2 Advanced Expert System™ (BioMérieux, Marcy l’Étoile, France) according to the manufacturer’s instructions [65].

Phenotypic detection of the virulence attributesDetermination of the biofilm-forming ability by MTP method

A 100 µL-suspension of each tested isolate, prepared to match the turbidity of a 0.5 M McFarland standard (1 × 106 to 5 × 106 CFU/mL), was used to inoculate a well of a 96-well microtiter plate containing 100 µL of double-strength RPMI 1640 broth supplemented with L-glutamine and phenol red (Merck, Darmstadt, Germany), without NaHCO3, and buffered with morpholinopropane sulfonic acid (MOPS, Merck, Darmstadt, Germany). Following 48 h of incubation at 37 °C, planktonic cells were removed, and the wells were washed twice. Biofilms were then stained with 0.2% crystal violet, solubilized in 95% ethanol, and the absorbance was measured at 630 nm. The optical density of each strain (ODs) was compared to the absorbance of the negative control (ODnc), containing 100 µL sterile saline instead of yeast inoculum. The results were interpreted as follows: no biofilm formation (ODs ≤ ODnc), weak biofilm formation (ODnc < ODs ≤ 2 ODnc), moderate biofilm formation (2 ODnc < ODs ≤ 4 ODnc), and strong biofilm formation (4 ODnc < ODs) [21]. All isolates were tested in triplicate. Quality controls, C. albicans ATCC 231GI and ATCC 10231 were included in the experiment.

Production of proteinase enzymes

The ability of the tested isolates to produce proteinase enzymes was assessed using the method described by Edward et al. with few modifications [21]. Bovine serum albumin (BSA) medium was prepared using 2% dextrose, 0.1% KH2PO4, 0.05% MgSO4, 2% agar, and 1% BSA (Himedia, Mumbai, India), then 20 µL of yeast cells suspension (at a density of 106 cells/mL) were dispensed into cups punched in the BSA medium. The plates were incubated at 37 °C for six days. The precipitation zone (Pz) value was calculated as the ratio of the diameter of the cup to the total diameter of the cup plus the precipitation zone, and the isolates were segregated accordingly into high producers (Pz = 0.35–0.5), moderate producers (Pz = 0.51–0.74), low producers (Pz = 0.75–0.9), and non-producers (Pz = 1). The test was performed in duplicate. C. albicans ATCC 231GI and ATCC 10231 were included in the experiment as quality controls.

Production of phospholipase enzymes

The egg yolk agar medium consisting of SDA, 1 M NaCl, 0.005 M CaCl2, and 8% sterile egg yolk emulsion (Himedia, Mumbai, India) was used to screen the isolates’ phospholipase activity. The egg yolk emulsion was centrifuged at 500 xg for 15 min at room temperature; the supernatant was completed to its initial volume with sterile DW and then added to the autoclaved medium. Twenty mL of the resultant medium were poured into each plate of 90 to 100 mm diameter. Aliquots of 20 µL of Candida suspension (approximately 106 CFU/mL) were introduced into cups previously punched in the medium. The plates were incubated at 37 °C for 48 h, the Pz value was computed as the ratio between the diameter of the cup and the total diameter of the cup plus the precipitation zone, and the results were interpreted as follows: Pz < 0.63 indicates a strong enzymatic activity, 0.63 < Pz < 1.0, a moderate one, while Pz = 1 refers to no enzyme activity [21]. The test was performed in duplicate. Quality controls, C. albicans ATCC 231GI and ATCC 10231 were included in the experiment.

Fluconazole susceptibility testing

The susceptibility of the isolates to FLC was determined using the DD method according to the performance standards for antimicrobial susceptibility testing of Clinical and Laboratory Standards Institute (CLSI) M44-Ed3 [66]. The FLC disks (25 µg, Himedia, Mumbai, India) were placed onto Müller-Hinton agar (Oxoid, Hampshire, UK) supplemented with 2% dextrose and 0.5 µg/mL methylene blue dye and inoculated with a suspension of yeast cells equivalent to 1 × 106 to 5 × 106 CFU/mL. The plates were incubated at 37 °C for 24–48 h. According to the diameters of the developed inhibition zones, Candida isolates were classified as S, SDD, or R following CLSI guidelines M27M44S-Ed3 [67]. Minimum inhibitory concentrations of FLC and repurposing agents: AK, COL, DEX, SMX, and KT were determined in the tested isolates using the BMD method in accordance with CLSI document M27-Ed4 [68]. A 50% inhibition in growth defined the MICs of FLC and KT [23, 67]. The MICs of DEX and SMX were indicated by 80% inhibition [20, 56], while 100% inhibition defined the MICs of AK and COL [21, 69]. Growth-positive and sterility control wells were included in each experiment, and C. albicans ATCC 231GI and ATCC 10231 were included as quality controls.

Molecular characterization of FLC resistance mechanismsQuantification of ERG11 and efflux pump genes using RT-PCR

The Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific Inc., Massachusetts, USA) was used to determine the expression levels of the ERG11 gene as well as those of the efflux pump genes (MDR1, CDR1, and CDR2) in triplicate. Initially, RNA was extracted using TRIzol® (Invitrogen™, Fischer Scientific, CA, USA) according to the manufacturer’s instructions, quantified by nanodrop Onec (Thermo Scientific, Massachusetts, USA), and then converted to cDNA using Topscript RT Drymix dN18/dN6 Kit (Enzynomics, Daejeon, Korea). Amplification of cDNA was carried out using primers obtained from Willowfort®, Birmingham, UK, for the efflux pump genes and from Eurofins genomics, Ebersberg, Germany, for the ERG11 gene (Table 5). The composition of the reaction mixture and the cycling conditions are presented in Additional file 1: Table S5. To ensure the lack of primer-dimers, melting curves analysis was performed at 95 °C for 15 s followed by 50 °C for 1 min in the case of MDR1, CDR1, and CDR2, or 55 °C for 1 min in the case of ERG11. A representative example of melting curve analysis performed for the CDR2 gene in C. albicans ATCC 10231 and 11 Candida spp. isolates is presented in Additional file 2: Fig. S3. The expression levels of all genes were normalized to the expression level of the housekeeping ACT1 gene and compared to the expression levels in the FLC-S C. albicans ATCC 10231 using the 2−ΔΔCt method.

Table 5 Primer pairs used for RT-PCR amplification and sequencing of the selected genes in this studyPCR amplification and sequencing of the ERG11 gene

For the PCR amplification of the ERG11 gene, DNA was extracted according to QIAamp® DNA Mini and Blood Mini (Qiagen, Hilden, Germany) Handbook with a single modification, in which the zymolase enzyme was replaced with 0.2 g of 0.5 mm glass beads, followed by vortexing for 15 min [72]. The DNA concentration and purity were assessed before the amplification of the full-length ERG11 gene (1587 bp), along with 71 bp upstream and 131 bp downstream of the gene using the forward and reverse primer pair indicated in Table 5. The composition of the reaction mixture and the cycling conditions are presented in Additional file 1: Table S5. The PCR product was electrophoresed on a 1% agarose gel to confirm that the amplicon was of the expected size through alignment with a 100 bp DNA ladder H3 RTU (Bio-helix Co. LTD, New Taipei City, Taiwan).

The PCR product was then purified according to the GeneJET Gel Extraction Kit’s (Thermo Scientific, MA, USA) manual and sequenced through primer elongation at three sites (forward, internal forward, and reverse primers, Table 5) using an ABI 3730xl sequencer (Thermo Fisher Scientific Inc., MA, USA) at GATC Biotech AG (Eurofins scientific, Cologne, Germany). The sequencing results were analyzed using BLAST® (National Center for Biotechnology Information, NCBI, MD, USA) and FinchTV 1.4.0 (Geospiza Inc., WA, USA). The translated sequence was then compared with that of a previously published ERG11 gene from an FLC-S strain available at NCBI (XM_002550939.1) using ProteinBLAST®. A representative example of the sequence analysis is shown in Additional file 2: Fig. S4.

In vitro assessment of FLC interaction with potential repurposing agentsCheckerboard titration technique

The interaction of FLC with each of the five repurposing agents was assessed using the checkerboard titration technique against representative Candida isolates as previously described by Eldesouky et al. [73]. Fluconazole was tested in two-fold dilutions over a range of 500 to 0.5 µg/mL with AK (range, 32768 to 256 µg/mL), COL (range, 4096 to 32 µg/mL), DEX (range, 1000 to 8 µg/mL), SMX (range, 4096 to 32 µg/mL), and KT (range, 3750 to 32 µg/mL). The interactions between the tested drugs were determined by calculating the fractional inhibitory concentration index (ƩFICI), interpreted as follows: synergism (Syn); ƩFICI ≤ 0.5, additivity (Add); ƩFICI > 0.5 to ≤ 1, and indifference (Ind); ƩFICI > 1 and ≤ 4.

Resistance modulation assay

To evaluate the FLC resistance-modifying activity of the five repurposing agents, the MF was calculated as the ratio between the MIC of FLC alone and its MIC in the presence of a sub-inhibitory concentration of each agent, equivalent to 0.25X MIC against the most sensitive tested isolate [74]. An MF > 2 was set as the cut-off indicating significant resistance modulation [75].

Rhodamine efflux assay

To investigate whether AK and DEX influence the activity of efflux pumps in Candida isolates, the rhodamine efflux assay was conducted according to Edward et al. [21]. Cells were grown in YPD broth, harvested by centrifugation (6000 rpm at 4 °C for 5 min), washed with glucose-free phosphate-buffered saline (PBS), and their count was adjusted to 1 × 107 cells/mL. Next, a 10 mM rhodamine solution (Loba Chemie, Mumbai, India) prepared in 95% ethanol was added to the Candida suspension to reach a final concentration of 10 µM [76]. The culture was incubated with rhodamine at 37 °C for 50 min and then kept on ice for 10 min. Cells were collected, washed with glucose-free PBS, and resuspended in 5% glucose/PBS. The tested drugs, AK and DEX, were added to reach a final sub-inhibitory concentration of 4096 and 250 µg/mL, respectively. A rhodamine-alone group (without drugs) served as a control. The fluorescence intensity of extracellular rhodamine was recorded by a spectrofluorometer (Shimadzu, Kyoto, Japan) at time intervals of 0, 30, 60, 90, and 120 min, with excitation at 485 nm and emission at 530 nm. For each result, an average value of three biological samples was used.

In vivo assessment of combined therapy

A murine model was employed to evaluate the efficacy of the suggested combinations (FLC combined with AK, COL, or DEX) in vivo. Five-week-old female Swiss albino mice (18–22 g) were housed in animal rooms maintained at 23 ± 2 °C with 50 ± 20% relative humidity. The mice were divided into groups where food and water were provided ad libitum.

Organ bioburden experiment

The bladder bioburden was determined as previously reported by Mohamed et al. with some modifications [77]. Four mice were allocated into each of the following groups: control, FLC, repurposing agent (AK, COL, or DEX), and the combination group. The mice were challenged IP with a 200 µL injection of C. albicans, CA9 isolate, equivalent to 1 × 108 to 3 × 108 CFU/mouse. The inoculum of CA9 was prepared from an overnight culture in 0.9% NaCl containing 5% gastric mucin (Oxoid, Hampshire, UK). One hour post-infection, the control group was injected IP with 200 µL water for injection (WFI), the FLC group received 50 mg/kg FLC, the repurposing agent group received 40 mg/kg AK, 0.6 mg/kg DEX, or 5 mg/kg COL, while the combination group was treated with both FLC and the repurposing agent at the above-mentioned doses. Mice were sacrificed by cervical dislocation 24 h after treatment administration, and the bladder was dissected and homogenized. The fungal count in the bladder was then determined following serial dilution and cultivation on SDA plates in duplicate.

Animal survival experiment

To further assess the in vivo efficacy of the FLC/COL combination, animal survival was monitored according to Mohamed et al. with few modifications [78]. Briefly, four groups each containing six mice were infected IP with a 200 µL injection of CA9 isolate suspended in 0.9% NaCl solution containing 5% mucin at a dose equivalent to 1.5X the minimal lethal dose (MLD). This inoculum killed 100% of the untreated mice within a maximum of 48 h post-infection. One hour post-infection, the first group received 200 µL WFI, the second was treated with 50 mg/kg FLC, the third was injected with 5 mg/kg COL, and the fourth received 50 mg/kg FLC and 5 mg/kg COL simultaneously. All doses were administered IP and were repeated at 25 and 49 h post-infection. The survival of challenged mice was then observed for 7 days, and deaths were recorded.

Histopathological examination

To examine the tissue-protective activity of the FLC/COL combination, 16 mice challenged IP with a 200 µL injection of CA9 isolate equivalent to 1.5X MLD were segregated into four groups. One hour post-infection, the first group received 200 µL WFI, the second received 50 mg/kg FLC, the third was administered 5 mg/kg COL, and the fourth was treated with both FLC and COL IP at the mentioned doses. Mice were sacrificed 24 h post-infection, and their bladders were harvested and fixed in 10% buffered formalin. Three 5-mm-thick serial sections were cut from the paraffin-embedded tissue and stained with hematoxylin and eosin. The degree of inflammation in each section was examined by a histopathologist blinded to the sample origin and graded according to the criteria established by Hopkins et al. [79]. Bladders isolated from uninfected mice were assessed for comparative purposes.

Statistical analysis

IBM® SPSS® statistics 25 program (IBM, NY, USA) was utilized to deduce the correlation between the resistance of the Candida isolates to FLC and their virulence, and the significance of such correlation through the calculation of rs and the p-value. Microsoft Excel Spreadsheet Software (Microsoft, WA, USA) was used to perform the t-test to statistically analyze the RT-PCR results and detect significance in the gene expression levels. The t-test was applied as well to assess the significance of the reduction in the fluorescence intensity upon treatment with AK or DEX in the rhodamine efflux assay. For the analysis of the animal survival results, Kaplan Meier survival analysis and Log-rank (Mantel-Cox) test were performed using GraphPad Prism 9.5.1 (GraphPad Software, CA, USA). A p-value < 0.05 indicated statistical significance.