Aspergillus fumigatus

Antibacterial activity was evaluated according to procedures previously described by [19 (link)] using three Gram-positive bacteria Bacillus cereus (human isolate), Staphylococcus aureus (ATCC 11632), and Listeria monicytogenes (NCTC 7973) and three Gram-negative bacteria Escherichia coli (ATCC 35210), Pseudomonas aeruginosa (ATCC 27853), and Salmonella typhimurium (ATCC 13311). The minimum inhibitory concentration (MIC) and minimum bactericidal (MBC) concentration were determined, and streptomycin and ampicillin were used as positive controls. On the other hand, the antifungal activity was evaluated following the protocol described by [44 (link)], using Aspergillus fumigatus (ATCC 1022), Aspergillus versicolor (ATCC 11730), Aspergillus niger (ATCC 6275), Penicillium funiculosum (ATCC 36839), Penicillium ochrachloron (ATCC 9112), and Penicillium verrucosum var. cyclopium (food isolate). The MIC and minimum fungicidal concentration (MFC) were determined. Ketokonazole and bifonazole were used as positive controls. The microorganisms were purchased at the Mycology Laboratory of the Department of Plant Physiology of the Institute for Biological Research “Siniša Stanković” at the University of Belgrade, Serbia.

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.

The antibacterial and antifungal activities of the extracts were determined according to methods described in detail by Pires et al. (2018) [35 (link)] and Heleno et al. (2013) [46 (link)], respectively. Five Gram-negative bacteria (Enterobacter cloacae—ATCC 49741, Escherichia coli—ATCC 25922, Pseudomonas aeruginosa—ATCC 9027, Salmonella enterica—ATCC 13076, and Yersinia enterocolitica—ATCC 8610) and three Gram-positive bacteria (Bacillus cereus—ATCC 11778, Listeria monocytogenes—ATCC 19111 and Staphylococcus aureus—ATCC 25923), besides two fungi strains (Aspergillus fumigatus—ATCC 204305 and Aspergillus brasiliensis—ATCC 16404) purchased from Frilabo (Porto, Portugal) were used. All work was performed with sterile materials handled under laminar flow. Prior to the assays, microorganisms were incubated (37 °C ± 0.5 °C for 24 h for bacteria and 25 °C ± 0.5 °C for 72 h for fungi) with appropriate media for each strain to reach a state of exponential growth. Negative controls of the extract and culture medium were prepared, and the antibiotics streptomycin, methicillin, ampicillin and antifungal ketoconazole served as positive controls. The antimicrobial potential was assessed as the minimum concentration required to inhibit the microorganism growth (MIB) and to cause bacteria or fungi death (MBC or MFC, respectively).

Several strains were used in the research: bacteria – Bacillus sp. (ATCC 51912), Enterobacter aerogenes (ATCC 29009), Enterococcus faecalis (ATCC 33186), Escherichia coli (ATCC 25922), Haemophilus influenzae (DSM 4690), Neisseria meningitidis (ATCC 53414), Proteus mirabilis (DSM 4479), Pseudomonas aeruginosa (DSM 13626), Serratia marcescens (DSM 50904), Staphylococcus aureus (ATCC 33497), Staphylococcus epidermidis (ATCC 35983), Staphylococcus haemolyticus (DSM 20263), Streptococcus agalactiae (DSM 2134), Streptococcus pneumoniae (ATCC 49619), Streptococcus pyogenes (DSM 20565), Streptococcus salivarius (DSM 20617), fungi – Aspergillus fumigatus (ATCC 14110), Candida albicans (ATCC 10231), Candida glabrata (DSM 11950), Candida parapsilosis (DSM 5784), Candida tropicalis (ATCC 20115).