Cryptococcus neoformans var. grubii strain H99 (71 (link)) and the cln1 mutant strain CNAG_06092, obtained from a collection of mutants deposited at the ATCC by H. D. Madhani (20 (link)), were used in this study. The strains were routinely grown in liquid Sabouraud medium (Oxoid, Ltd., Basingstoke, Hampshire, England) at 30°C or 37°C with moderate shaking (150 rpm). To induce capsule growth, the cells were transferred to 10% Sabouraud medium buffered at pH 7.3 with 50 mM MOPS (morpholinepropanesulfonic acid) buffer (Sigma-Aldrich, St. Louis, MO) at 30°C or 37°C with shaking (12 (link)). Murine macrophage-like cells (72 (link)) were grown in feeding medium, which contains Dulbecco’s modified Eagle’s medium (Lonza, Verbiers, Belgium) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (HyClone-Perbio), 10% NCTC medium (Sigma-Aldrich, Steinheim, Germany) and 1% nonessential amino acids (Sigma-Aldrich, Steinheim, Germany). Macrophages were regularly maintained at 37°C in a 5% CO2-enriched atmosphere.
Sabouraud medium
Manufactured by Merck Group
Sourced in Germany
Sabouraud medium is a culture medium used for the isolation and cultivation of pathogenic fungi. It is a nutrient-rich agar-based medium that supports the growth of a wide range of fungal species. The medium contains glucose, peptone, and other essential nutrients required for fungal growth.
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Lab products found in correlation
Non essential amino acids (neaa), by Merck Group (1 mentions)
Sabouraud medium, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Lonza (1 mentions)
Voriconazole vcz, by Merck Group (1 mentions)
Tsa medium, by Merck Group (1 mentions)
Mas 100 eco air sampler, by Merck Group (1 mentions)
Malt extract agar, by Merck Group (1 mentions)
Bigdye terminator sequencing kit, by Thermo Fisher Scientific (1 mentions)
Exosap it technique, by GE Healthcare (1 mentions)
Abi 3500 3500xl, by Thermo Fisher Scientific (1 mentions)
Abi sequence scanner v 1, by Thermo Fisher Scientific (1 mentions)
Seq agencourt, by Beckman Coulter (1 mentions)
8 protocols using sabouraud medium
1
Cryptococcus neoformans Capsule Induction
2
Antifungal Activity of Peptides Against Candida albicans
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Candida albicans (ATCC© 10231TM) was cultured in Sabouraud medium (Sigma-Aldrich) supplemented with tetracycline (10 μg/mL) and cefotaxime (10 μg/mL) at 37 °C for 24 h. Candida albicans (OD600nm = 0,001) were plated in 96-well plates and treated either with different concentrations of the peptides of interest, and/or with different concentrations of voriconazole (VCZ) (Sigma-Aldrich). As a positive control, cells were treated with 10 μg/mL VCZ. After 24 hours incubation, yeast growth was assessed by optical density OD600nm using a spectrophotometer (Multiscan EX).
The MIC of the antifungal agents, defined as the lowest concentration of drug able to inhibit 100% of the growth of a pathogen was determined using a modified Gompertz model 47 (link).
The efficiency of the combination between D-Ctl and VCZ was determined by the calculation of the Fractional inhibitory concentration index (FICI) according to the following formula: FICI = FICD-Ctl + FICVCZ = (MICD-Ctl in combination/MICD-Ctl + (MICVCZ in combination/MICVCZ . According to EUCAST38 , the effect of the combination is synergistic if FICI ≤ 0,5, additive if 0,5 < FICI ≤ 1, indifferent if 1 < FICI < 2 and antagonistic if FICI ≥ 2.
The MIC of the antifungal agents, defined as the lowest concentration of drug able to inhibit 100% of the growth of a pathogen was determined using a modified Gompertz model 47 (link).
The efficiency of the combination between D-Ctl and VCZ was determined by the calculation of the Fractional inhibitory concentration index (FICI) according to the following formula: FICI = FICD-Ctl + FICVCZ = (MICD-Ctl in combination/MICD-Ctl + (MICVCZ in combination/MICVCZ . According to EUCAST38 , the effect of the combination is synergistic if FICI ≤ 0,5, additive if 0,5 < FICI ≤ 1, indifferent if 1 < FICI < 2 and antagonistic if FICI ≥ 2.
3
M. audouinii Identification Protocol
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All samples were confirmed as M. audouinii by conventional identification. This was based on colony morphology and microscopic examination. All strains were subcultivated on Malt agar at 30°C (Fisher Scientific, Gent, Belgium). After confirmation of the identification, the strains were transferred onto liquid Sabouraud medium (Sigma-Aldrich, Diegem, Belgium) and grown for 7-10 days at 30°C before DNA extraction.
4
Fungal Degradation of 4-n-Nonylphenol
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The following species of genus Metarhizium were cultured: M. acridum ARSEF7486, M. anisopliae ARSEF7487, M. brunneum ARSEF2107, M. globosum ARSEF2596, M. guizhouense ARSEF6238, M. lepidiotae ARSEF7412, M. majus ARSEF1914, and M. robertsii ARSEF727. These strains were obtained from the Collection of Entomopathogenic Fungal Cultures (ARSEF, USA). The cultures were maintained on ZT agar slants for 14 days and were inoculated in 20 mL Sabouraud medium (Sigma-Aldrich, Germany) in 100 mL Erlenmeyer flasks. After 1 d of incubation, the precultures were transferred to a fresh minimal medium at a ratio of 1:9 (Różalska et al., 2015a) . The concentration of 4-n-NP in the culture medium was 50 mg L -1 . Stock solution was prepared in ethanol at a concentration of 20 mg mL -1 . Abiotic controls (without fungal biomass) and biotic controls (without toxic substrate) were also prepared. The samples were incubated on a rotary shaker (160 rpm) at 28 C. For the estimation of fungal biomass and for gas chromatography-mass spectrometry (GC-MS) analyses, samples were collected at 6-hour intervals. For biomass estimation the mycelia were filtered through a Whatman filter paper number 1 (Sigma-Aldrich, Germany) and then dried at 105 °C until a constant weight was obtained. Other chemicals and reagents were purchased from Sigma-Aldrich (Germany) unless otherwise stated.
5
Microbial Contamination Assessment of Surfaces
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For microbiological analyses, sampling was performed in duplicate by using Replicate Organism Detection and Counting (RODAC) contact plates containing the following specific culture media: total bacteria (TSA medium, Merck Millipore, Milan, Italy), Staphylococcus spp. (Baird Parker medium, Merck Millipore, Milan, Italy), Enterobacteriaceae spp. (MacConkey medium, Merck Millipore, Milan, Italy), Acinetobacter spp. (Herella medium, Lickson, Milan, Italy), Pseudomonas spp. (Cetrimide medium, Incofar, Modena, Italy), Clostridium difficile (Clostridium difficile selective medium, Lickson, Milan, Italy), Enterococcus spp. (BEA medium, Incofar, Modena, Italy) and mycetes (Sabouraud medium, Merck Millipore, Milan, Italy). Plates for general or selective growth of bacteria were incubated at 30 °C for 24–48 h (respectively for general and selective media), whereas plates for the specific growth of mycetes were incubated at 25 °C for 72 h. Colony forming units (CFU) were then enumerated. Plates containing ≥ 200 CFUs were considered to have 200 CFUs, following the guideline INAIL-2017 [28 ]. A total of 216 samples were collected from surfaces.
6
Quantitative Analysis of Molds in Diverse Workplaces
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Quantitative analysis of molds were undertaken in the following working environments: archive (N = 1), museum (N = 1), libraries (N = 2), tannery (N = 1) and composting plant (N = 1). Descriptions of the working environments, and the number of air and surface samples are given in Table 1 .
Airborne molds were isolated using an MAS-100 Eco Air Sampler (Merck, Darmstadt, Germany) according to the PN-EN 13098:2007 standard. Fifty and 100 L air samples were taken on DG18 agar medium (Dichloran Glicerol Selective Medium, Merck) and MEA medium (Malt Extract Agar, Merck) with chloramphenicol (0.1%) for determining total fungal number (including xerophilic and hydrophilic molds). Samples from surfaces (production surfaces, machinery and equipment) were collected using Replicate Organism Detection And Counting (RODAC) Envirocheck® plates (Merck) containing Sabouraud medium (Merck).
The samples were incubated at 27 ± 2 °C for 5 days. Following incubation colonies were counted, and the results were expressed in CFU/m3 (air) or CFU/100 cm2 (surfaces). The proportion of each Alternaria isolate in the pool of molds was determined, and the incidence of airborne and surface molds in each workplace was measured.
Airborne molds were isolated using an MAS-100 Eco Air Sampler (Merck, Darmstadt, Germany) according to the PN-EN 13098:2007 standard. Fifty and 100 L air samples were taken on DG18 agar medium (Dichloran Glicerol Selective Medium, Merck) and MEA medium (Malt Extract Agar, Merck) with chloramphenicol (0.1%) for determining total fungal number (including xerophilic and hydrophilic molds). Samples from surfaces (production surfaces, machinery and equipment) were collected using Replicate Organism Detection And Counting (RODAC) Envirocheck® plates (Merck) containing Sabouraud medium (Merck).
The samples were incubated at 27 ± 2 °C for 5 days. Following incubation colonies were counted, and the results were expressed in CFU/m3 (air) or CFU/100 cm2 (surfaces). The proportion of each Alternaria isolate in the pool of molds was determined, and the incidence of airborne and surface molds in each workplace was measured.
7
Antagonistic Activity of MGMM1 Against Phytopathogens
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The antagonistic activity of MGMM1 against F. oxysporum f. sp. radicis-lycopersici (Forl) ZUM2407, Alternaria alternata, F. graminearum and F. spp., was carried out using a dual culture (confrontation) assay method on Sabouraud medium (Merck, Darmstadt, Germany). The cell suspension of MGMM1 obtained in Section 2.1 was used as the inoculum.
8
Fungal SQLE gene sequencing protocol
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Total DNA was extracted from fresh fungal cultures growing in liquid Sabouraud medium (Merck, Darmstadt, Germany) using the Maxwell 16 Cell SEV kit (Promega, USA). The PCR amplifying the SQLE gene was done as described by Yamada et al. [17 (link)]. Purification of PCR products was then performed using the Exosap IT technique (Amersham, GE Healthcare Europe GmbH, Belgium). Bidirectional sequence data were generated after purification using the BigDye terminator sequencing kit (Applied Biosystems, Life Technologies, Merelbeke, Belgium). Sequenced products were finally purified using the kit clean Seq Agencourt (Beckman Coulter Life Science, Villepinte, France). The sequencing was done on the automate ABI 3500/3500XL (Applied Biosystems, Life Technologies, Belgium). Sequences were edited using the ABI Sequence Scanner V.1.0 software (Applied Biosystems, Life Technologies, Merelbeke, Belgium). Forward and reverse sequences generated by the software were then blasted, and the consensus alignment was transduced in Expasy portal (SIB Bioinformatics Resource Portal, Lausanne, Switzerland) on amino acid sequence. The delivery sequence was then compared to the reference SQLE amino-acid sequence of T. mentagrophytes GenBank: BAL48859.1.
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