C tropicalis

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.

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.

Compounds 14 and 15 were evaluated for antifungal activity in vitro using commercial antifungals such as fluconazole for yeasts and itraconazole for fungi. Activities are reported in the minimum inhibitory concentration (MIC) values using the serial microdilution method on 96-well plates. The following reference strains were purchased from the American Type Culture Collection (ATCC): Candida albicans ATCC 24433, C. glabrata ATCC 66032, C. guilliermondii ATCC 6260, C. krusei ATCC 14243, C. tropicalis ATCC 750, A. niger ATCC 16404 and A. fumigatus ATCC 204305. The MIC values were determined according to the M27-S4 method of the Clinical Laboratory Standards Institute for yeasts [34 ] and the M38 method for fungi [35 ]. The RPMI-1740 (Sigma) culture medium buffered with 0.165 M of HEPES (Sigma) was used. The MIC values were defined as the minimum concentration that inhibits growth of ≥ 50% Candida spp. and % Aspergillus spp. in comparison to the controls. The compounds were dissolved in ethanol and serially diluted in the growth medium. The yeasts were incubated to 35–37 °C and the MIC values were measured at 24 and 48 h; the fungi were incubated to 35–37 °C and measured at 48 and 72 h.

The microorganisms used in this study were Candida albicans ATCC 9002, C. parapsilosis ATCC 22019, C. tropicalis ATCC 750, C. krusei ATCC 6258, C. lusitaniae ATCC 200950, (American Type Culture Collection), Cryptococcus neoformans IP95026 (Institut Pasteur France) and two clinical isolates namely C. guilliermondii and C. glabrata (Centre Pasteur Yaounde-Cameroon). Sabouraud dextrose agar (SDA) (Liofilchem Laboratories) was used for the maintenance and culture of fungal strains while Sabouraud dextrose broth (SDB) was used for the determination of the minimum inhibitory concentrations (MICs) and the minimum fungicidal concentrations (MFCs).