C parapsilosis

Antifungal evaluation was performed using a microdilution broth method [29 (link)] against eight fungal strains from the Czech Collection of Microorganisms (CCM) (Candida albicans CCM 8320 (ATCC 24433), C. krusei CCM 8271 (ATCC 6258), C. parapsilosis CCM 8260 (ATCC 22019), C. tropicalis CCM 8264 (ATCC 750), Aspergillus flavus CCM 8363, Absidia/Lichtheimia corymbifera CCM 8077 and Trichophyton interdigitale CCM 8377 (ATCC 9533) or the American Type Collection Cultures (ATCC, Mannasas, VA, USA) (Aspergillus fumigatus ATCC 204305). Compounds were dissolved in DMSO and diluted in a twofold manner with RPMI 1640 medium, with glutamine and 2% glucose, buffered to pH 7.0 (3-morpholinopropane-1-sulfonic acid). The final concentration of DMSO in the tested medium did not exceed 2.5% (v/v) of the total solution composition. Static incubation was performed in the dark and in humid atmosphere, at 35 °C, for 24 and 48 h (72 and 120 h for Trichophyton interdigitale respectively). Drug-free controls were included. MIC was inspected visually or making use of Alamar Blue staining. The standards were amphotericin B and fluconazole. All experiments were conducted in duplicate. For the results to be valid, the difference in MIC for one compound determined from two parallel measurements must not be greater than one step on the dilution scale.

To establish diagnostic sensitivity and specificity, heterologous control strains were selected based on clinical similarity to H. capsulatum, and consisted of P. brasiliensis 18 and B-339 (ATCC 32069), Candida albicans, C. parapsilosis, C. neoformans ATCC 24067, and Aspergillus spp., all of which were obtained from Micoteca do Instituto de Medicina Tropical de Sao Paulo. Positive control strains consisted of H. capsulatum ATCC 28308 (CDC: B973), ATCC 12700 (CDC: A811), and HC200 (GenBank: DQ239887).

The following standard strains were used: C. albicans (ATCC 90028), C. dubliniensis (ATCC MYA-646), C. guilliermondii (ATCC 6260), C. glabrata (ATCC 2001), C. krusei (ATCC 6258), C. metapsilosis (ATCC 96143), C. orthopsilosis (ATCC 96141), C. parapsilosis (ATCC 22019) and C. tropicalis (ATCC 13803), obtained from American Type Culture Collection (ATCC). In addition, a clinical isolate of C. auris was used, kindly provided by Hospital das Clinicas, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo (HCFMRP/USP), isolated from the blood of a patient. All yeasts used in this study are part of the culture collection of the Laboratory of Antimicrobial Testing of the Federal University of Uberlândia (LEA/UFU), preserved in deep freezing at − 80 °C until the start of tests.

Eight strains of yeast were obtained from the American Type Culture Collection (ATCC): C. albicans ATCC 10231, C. glabrata ATCC 15126, C. parapsilosis ATCC 22099, C. tropicalis ATCC 13803, C. krusei ATCC 14243, Candida kefyr ATCC 204093, C. auris ATCC MYA-5001, and G. capitatum ATCC 201230. The clinical isolates of C. albicans (CA1-CA20) were obtained as stock cultures from the Jan Boży Independent Public Provincial Hospital in Lublin, Poland. The strains were identified using VITEK 2 YST IC CARDS (Biomerieux). Stock cultures were stored at −70°C, subcultured on YPDA medium (1% yeast extract, 2% peptone, 2% dextrose, and 2% agar), and stored at 4°C. The fungal strain cultures were routinely maintained in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) at 30°C.

C. albicans SC5314 (ATCC MYA-2876), C. albicans CGMCC 2.2086, C. parapsilosis (ATCC 22019), Cryptococcus neoformans (ATCC 208821), and Rhizoctonia solani (CGMCC 3.7376) were obtained from China General Microbiological Culture Collection Center. Aspergillus niger (CCTCC AF 93021), A. fumigatus (CCTCC AF 93048), A. alternata (CCTCC AF 93103), F. oxysporum (CCTCC AF 93247), and B. cinerea (CCTCC AF 93110) were obtained from China Center for Type Culture Collection. All strains were maintained on YPD or YPDA^{33 (link)}. Hyphal development was induced using the yeast carbon base/fetal bovine serum (YCB/FBS) liquid media, containing 1.17% (w/v) yeast carbon base (BD Biosciences), 1% (w/v) glucose, and 10% (v/v) fetal bovine serum (Gibco BRL, USA).

All chemicals (reagents, standards, ELISA kit, and culture media) were procured from Sigma-Aldrich (St. Louis, MO, USA), R&D Systems (Minneapolis, MN, USA), and Promega Corporation (Madison, WI, USA). The following standard bacterial and yeast strains were used in the microbiological assays: Methicillin-resistant Staphylococcus aureus (MRSA, ATCC 33591), Staphylococcus aureus (ATCC 25923), Klebsiella pneumoniae (ATCC 27736), Escherichia coli EHEC (ATCC 43895), Pseudomonas aeruginosa (ATCC 27853), Candida albicans (MYA 2876), C. glabrata (ATCC 90030), C. tropicalis (ATCC 750), C. parapsilosis (ATCC 2019), Streptococcus salivarius (ATCC 7073), and S. sanguinis (SK36). The strains were grown in Sabouraud Dextrose agar (SDA, Kasvi), Brain Heart Infusion (BHI), or RPMI culture media.

C. albicans (catalog no. 90028), C. glabrata (catalog no. 90030), C. parapsilosis (catalog no. 90018) and S. aureus (catalog no. 29213) were purchased from ATCC. E. coli (bank no. 0077) were provided by the Antibiotic Resistant Isolate Bank of Centers for Disease Control and Prevention (CDC). The three Candida species were grown on YM agar/broth (catalog no. 271120, BD Biosciences). S. aureus was grown on Tryptic Soy agar/broth (catalog no. 211825, BD Biosciences). E coli was grown on LB agar/broth (1% w/v Tryptone, 0.5% w/v Yeast extract, 0.5% w/v NaCl, 0.1% v/v 1 N NaOH). For spiking experiments, a single colony of bacteria or yeast was inoculated into 2 mL of the respective broth and incubated overnight in a shaker incubator at 250 rpm at 37 °C (for bacteria) or 30 °C (for yeast). After overnight culturing, cells were spun down and resuspend in 10 mM Tris (pH 8.0). Optical density of the cells was then measured using NanoDrop 2000c at the wavelength of 600 nm. As described in the next section, the measured optical density (OD) of the cells was used to determine the concentration of the cells (i.e., CFU mL⁻¹) using a conversion factor obtained for each species.