Sampling and isolation of fungal elements
One hundred eighteen samples were collected from private laboratory and private hospitals in Egypt from five sections of immunocompromised patients including 50 diabetic patients, 29 pregnant women, 5 patients underwent organ transplantation (liver And kidney), 19 cancer patients and 15 burned and wounded patients. The collected samples included 59 urine samples, 39 oral swabs, 5 vaginal swabs and 15 skin swabs. All the collected samples were subjected to fungal enrichment by inoculation into Sabouraud dextrose broth (SDB) tubes, then the tubes were incubated at 30 °C for 24 h and then examined visually for turbidity.
Phenotypic identification of the isolates
Turbid SDB tubes suspected to contain fungal elements were identified by morphotyping, macroscopic and microscopic examination according to Montes et al. (2019).
Macroscopic identification
A loopful taken from every turbid SDB tube was streaked on Sabouraud dextrose agar (SDA, Lot. no 1594522/Oxoid) with chloramphenicol (16 mg/ml, Neo Quimica) (Marinho et al. 2010). Then the plates were incubated at 30 °C for 48 h. Each grown colony was checked for size, colour and shape.
One single colony grown from each SDA plate was picked up and streaked on CHROMagar (CONDA, Spain) plate, and then, the plates were incubated at 37 °C for 48 h. Green colonies are interpreted as C. albicans, blue colonies are defined as C. tropicalis, and light white to purple colonies are defined as C. glabrata; purple to pink colonies are defined as C. krusei; and pale colonies are referred to C. parapsilosis.
Microscopic identification
Gram staining
One single colony from each SDA plate was streaked on a clean glass slide, stained with Gram stain and examined microscopically under oil immersion lens.
Germ-tube test
One single colony from each SDA plate was picked up and incubated with 0.5 ml human serum in an Eppendorf at 37 °C for 2–3 h. After incubation, microscopic examination of a loopful from each Eppendorf was carried out (Souza 1998).
Chlamydospores forming test:
The test was performed using rice extract agar medium (REA) (10 g rice, 10 g bacteriological agar, and distilled water adjusted to a final volume of 1000 ml) supplemented with 8 ml of Tween 80 (Montes et al. 2019).
One single colony from SDA plate was streaked (very thinly) on the surface of (REA) plate in 3–4 broad zigzag lines then covered with cover glass and incubated for 9 h at 22–25 °C and then the plate was examined microscopically.
Biotyping
Biochemical identification of the Candida isolates was carried out using API 20C Aux strips. One single colony from young Candida isolate culture taken from SDA plate was immersed into an API 20C suspension tube, and the degree of turbidity was adjusted equal to 2Mcfirland tube. Suspension tube (100 μl) was added to API 20 C medium tube. The cupules of the strip were filled with the suspension from API 20C medium tube, and then, the strips were incubated at 30 °C (for 24, 48 and 72 h). After incubation, the turbidity of cupules was observed and recorded, then a profile number is generated. The obtained figures were subjected to computerized analysis to identify the Candida isolate to species level.
(DNA-based assay; nucleic acid-based assay) genotyping
DNA extraction
DNA extraction of yeast cells was carried out using mini-preparation procedure. To a 1.5-ml Eppendorf tube containing 500 μl of lysis buffer (400 mM Tris–HCl [pH 8.0], 60 mM EDTA [pH 8.0], 150 mM NaCl and 1% sodium dodecyl sulphate), a loopful of yeast colony was added aseptically by using a sterile loop, the tube was then left at room temperature for 10 min. After adding 150 µl of potassium acetate, pH 4.8 (5 M potassium acetate 60 ml, glacial acetic acid 11.5 ml, distilled water 28.5 ml), the tube was vortexed briefly and then centrifuged at > 10,000g for 1 min to remove the cellular debris and precipitated proteins. The supernatant was transferred to another 1.5-ml Eppendorf tube and centrifuged again as above. After transferring the supernatant to a new 1.5-ml Eppendorf tube, an equal volume of isopropyl alcohol was added. The tube was mixed briefly by inversion, centrifuged at > 10,000g for 2 min, and the supernatant was discarded. The resultant DNA pellet was washed in 300 µl ethanol 70% v/v. After centrifuging at 10, 000 g for 1 min, the supernatant was discarded (Lliu et al. 2002). The DNA pellet was added to EZ-10 Spin Columns (Bio Basic Inc.) and centrifuged at > 10,000 g for 10 min. DNA was eluted in 50 µl of 1X TE buffer and stored at − 20 °C.
PCR amplification
The PCR was used to amplify intergenic spacer regions; intertranscribed spacer (ITS) of gene encoding 5.8 S rDNA with primers ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3') (Willowfort.co.UK.). A reaction volume of 50 μl contained 25 μl master mix PCR [0.16 mM each deoxyribonucleoside triphosphate, 5 μl Taq DNA polymerase buffer, 2.5U Taq DNA-dependent DNA polymerase (intron master mix (i-Taq ™)), 0.2 μM each primer and 1 µg genomic DNA as a template. Reaction mixtures were subjected to initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation at 95 °C for 1 min, primer annealing at 50 °C for 1 min and elongation for 2 min at 72 °C. Final extension step was performed at 72 °C for 10 min. Negative control was carried out using sterile deionized water instead of template DNA. Red safe stained agarose gel (1.5%) was prepared in 1X TBE buffer (Tris base/boric acid/EDTA). Agarose gel electrophoresis was run at 100 V and the resulting bands were visualized by UV illumination. PCR products were stored at − 20 °C until used (Elena et al. 2015).
Antifungal activity of zinc oxide nanoparticles and commercial antibiotics against Candida isolates
Two commercially available antifungals (nystatin 100 unite/disc—Oxoid) and (Voriconazole 1 µg/disc—Oxoid) and zinc oxide nanoparticles (ZnONPs) used in this study are chemically synthesized and taken from Mona Mohammed Hassan a researcher at national Research Center.
Candida species selected for this test were cultivated on SDA and incubated at 37 °C for 24 h and a yeast suspension in distilled water equal to 1/2 McFarland was done.
A colloidal solution from 10 mg of ZnONPs dissolved in 500 μl distilled water was performed using a sonicator for 5 min. Three different ZnONPs concentrations (25–50–100 µg/disc) were prepared and the yeast suspension was spread on the SDA plate then the discs loaded by ZnONPs were distributed on the plate. The plates were incubated at 37 °C for 48 h. The inhibitory zone (s) was measured after the incubation period and recorded.
