Sabouraud agar

The MICs of crude extracts were determined against yeasts using broth microdilution techniques, according to the method described by CLSI [36 ]. MICs were determined in RPMI 1640 medium (Sigma, R6504), pH 7.0. The starting inoculum was 1.0 × 10⁶ CFU/mL. Microtiter plates were incubated at 37°C in a dark humid chamber, and MICs were recorded after 48 h. The lyophilized hydroethanolic extracts from endophytes were diluted in dimethylsulfoxide (Sigma, D8418), filtered in sterile membranes, and tested at 1000, 500, 250, 125, 62.5, 31.25, 7.8, 3.9, and 1.9 μg/mL concentrations. The MIC was defined as the lowest concentration of compounds without microorganism growth. Nystatin (Sigma, N6261) was used as the drug control. The minimal fungicidal concentrations (MFCs) were obtained from the MIC results. For this, an aliquot of 10 μL from the wells that showed inhibition was spread in Petri dishes with Sabouraud agar (Merck, 105438) and incubated at 37°C for 24 h. The MFCs were defined as the lowest concentration of crude extract that resulted in no growth when the treated culture was spread on plates with antibiotic-free medium after the incubation. All tests were repeated and confirmed.

This work was done using the reference strain H. pylori J99 (ATCC 700824, urease+cagA+vacA+, isolated from a duodenal ulcer) and the C. albicans ATCC 90028 strain. H. pylori J99, used as a representative of the species H. pylori, was cultured in plates containing Columbia agar (OXOID, United Kingdom) supplemented with 5% horse blood and DENT (OXOID, Basingstoke, United Kingdom) at 37 °C for 72 h in an incubator (Thermo Scientific, Waltham, MA, USA) under microaerobic conditions (10% CO₂). C. albicans ATCC 90028 was used as a representative of the genus Candida. It was cultured on Sabouraud agar (Merck, Darmstadt, Germany) supplemented with chloramphenicol (OXOID, Basingstoke, United Kingdom) and incubated at 37 °C for 24 h in an incubator (ZHICHENG, Shanghai, China) under aerobic conditions. H. pylori J99 strain is part of the culture collection of the Laboratory of Bacterial Pathogenicity of the University of Concepcion, Chile, the premises where this work was done. C. albicans ATCC 90028 was donated by Dr. Patricio Godoy, Institute of Clinical Microbiology, Universidad Austral de Chile, Valdivia, Chile.

Capsicum annuum L. fruits (accession UENF1381) were provided by the Laboratório de Melhoramento Genético Vegetal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes, Rio de Janeiro, Brazil.
The yeasts C. tropicalis (CE017) and Saccharomyces cerevisiae (1038) were obtained from the Departamento de Biologia, Universidade Federal do Ceará, Fortaleza, Brazil. Yeasts were maintained on Sabouraud agar (1% peptone, 2% glucose, and 1.7% agar-agar) (Merck).

During the 48 h incubation of the co-cultures, a 20 µL aliquot of each of them was obtained at times 0, 1, 3, 6, 12, 24 and 48 h and placed on a glass slide to prepare wet mounts for observation under an optical microscope, using the oil immersion 100× objective lens, (Leica, Wetzlar, Germany fitted with camera). For each co-culture, 200 yeast cells were counted and the percentage of yeast cells containing mobile bacteria-like bodies (Y-BLBs) was calculated. Additionally, to be used in the following assays, a sample from the co-cultures was added to 1 mL BB-5%FBS, vortexed (DLAB, Ontario, CA, USA) and 0.015 µg/mL clarithromycin (Sigma-Aldrich, St. Louis, MO, USA) added. This mixture was incubated under microaerobic conditions at 37 °C for 24 h to eliminate extracellular remaining H. pylori cells. Then, 20 µL of the culture were seeded on the surface of dishes containing Sabouraud agar (Merck, Darmstadt, Germany) plus 50 mg/L chloramphenicol (OXOID, Basingstoke, UK) (SA-CHL) and incubated for further 24 h at 37 °C under microaerobic conditions and colonies reseeded 6 times in SA-CHL to eliminate any residual extracellular H. pylori cells.

Fungal macroscopic parameters (colony color, texture, reverse color, border type) and colony diameters were observed in three different media. Colors follow the specification proposed by the OACC [23 ]. All isolates were inoculated in the following media: PDA, Sabouraud Agar (Merck^® KGaA, Darmstadt, Germany), and Grass Extract Dextrose Agar (GE). The Grass Extract Dextrose Agar was obtained by grinding 100 g of fresh leaves of Deschampsia antarctica into 400 mL of distilled water. Then the extract was filtered, 20 mL of its content was added to 100 mL of culture medium, with 10 g of dextrose and 12 g of agar added (quantities for one liter). The final volume was sterilized at 121 °C for 20 min and poured into sterile Petri dishes [24 (link)]. All media were incubated at 5, 10, 20, and 23.5 ± 1 °C. The morphological and microscopic characteristics were evaluated from 15–30 days. Media under the same conditions were used to determine the microscopic characters (hyphae, conidiophores, chlamydospores and conidia), and measures were obtained by determining the length/width of individual characters. All analyses are made under slides in light microscopy under 100× oil immersion objective and imaging was performed on a Zeiss Axio Imager A2 (Carl Zeiss, Oberkochen, Germany) equipped with Axiocam MRc system (Carl Zeiss, Oberkochen, Germany).

Fusarium oxysporum (5845), Fusarium solani (4014), Colletotrichum lindemuthianum (5771) and Colletotrichum gloeosporioides (5522) phytopathogenic fungi were maintained and cultured in Sabouraud agar (10 g/l peptone, 2 g/l glucose, 20 g/l agar) (Merck Millipore Brazil) in the LFBM, at the Centro de Biociências e Biotecnologia (CBB), UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil.

Following microorganism growth identification by the BacT/ALERT^® 3D system, blood culture media were collected from each bottle and subjected to Gram staining. Then, samples were subcultured on solid growth media, including blood agar (bioMérieux) and Sabouraud agar (Merck, Germany), and incubated at 35°C for 18–24 hours. To estimate the cell numbers in the bottles, 5 positive blood culture bottles were randomly selected. Then, 1 mL sample was aspirated from each of these bottles, serially 10‐fold diluted with sterile saline, and 50 μL of suspensions was plotted on the Sabouraud agar plate, and colonies were counted after 24 h of incubation (ranged from 7 × 10⁵ to 5 × 10⁷ CFU/mL). The rapid disc diffusion method was performed according to the RAST methodology standardized by the European Committee on Antimicrobial Susceptibility Testing. Following incubation, isolated colonies were subjected to analysis by the MALDI-TOF VITEK MS^® 3.0 system (bioMérieux, France) according to the manufacturer's instructions. Fluconazole susceptibility was assessed using a disk diffusion method according to the CLSI M44-A2 guidelines and a broth microdilution method according to the European Committee on Antimicrobial Susceptibility Testing guidelines [8 , 9 ].