Sabouraud dextrose agar

Vaginal swabs were inoculated on Sabouraud Dextrose Agar (SDA) (Liofilchem, Italy) for fungi isolation and incubated at 37°C for 48h. Direct wet mount examination and Gram staining were performed for all suspected colonies, and isolates were recultured on SDA for 48h at 35°C for purity purposes. Positive Candida cultures were further identified to the species level by the Chromatic Candida medium (Liofilchem, Italy) that allows the selective isolation and differentiation of Candida spp. based on colony color and morphology. The chromatic agar has been well documented in previous studies as for its high sensitivity and specificity for the identification of the most commonly encountered Candida spp. [13 (link)–15 (link)]. After 72h of incubation of Chromatic Candida plates at 37°C, green colonies were identified as C. albicans, creamy colored colonies were regarded as C. glabrata, and C. krusei colonies were pink with a whitish border. C. albicans isolates were further confirmed by germ tube test.

Each sourdough sample (25 g) was mixed with 225 mL of sterile saline water (0.9% NaCl w/v) in a stomacher bag and homogenized for 4 min using a Stomacher (Interscience). Ten-fold serial dilutions were made and plated on the following agar media and conditions: Plate Count Agar (PCA) (Biolife Italiana srl, Milan, Italy), incubated for 48–72 h at 30 ± 2 °C, for total mesophilic bacteria counts; Slanetz Bartley Agar (SLA) (Biolife Italiana srl), incubated for 24–48 h at 37 °C, for enterococci enumeration; Violet Red Bile Glucose Agar (VRBGA) (Biolife Italiana srl), anaerobically incubated at 37 °C for 24–48 h for Enterobacteria; Brilliance E. coli (BEC) (Oxoid, Milan, Italy), incubated at 37 °C for 18–24 h for Escherichia coli; Mannitol Salt Agar (MSA) (Biolife Italiana srl), incubated at 32 °C for 48 h, for staphylococci; modified MRS (mMRS), according to Corsetti and co-workers [13 (link)], anaerobically incubated at 30 ± 2 °C for 48 h, for lactic acid bacteria; Sabouraud Dextrose Agar (SDA) (Liofilchem, Roseto degli Abruzzi, Italy) and Wallerstein Laboratory Nutrient Agar (WLN) (Liofilchem), supplemented with chloramphenicol (100 mg/L), incubated at 30 ± 2 °C for 48–72 h, for yeasts count.

All microorganisms used in this study are listed in Table 1. Cell stocks were kept at −80 °C and subcultured once before the experiments. Filamentous fungi strains were maintained in Sabouraud Dextrose Agar (SDA) (Liofilchem s.r.l., Roseto D.A., Italy) or Potato Dextrose Agar (PDA) (Liofilchem s.r.l., Roseto D.A., Italy) for approximately 7 days at room temperature. For each experiment, conidia were harvested by flooding the agar surface with sterilized saline solution containing NaCl 8.00 g·L⁻¹ (Sigma-Aldrich, Sintra, Portugal), KCl 0.2 g·L⁻¹, Na₂HPO₄·2H₂O 1.44 g·L⁻¹, and KH₂PO₄ 0.24g·L⁻¹ (all from José Manuel Vaz Pereira, Benavente, Portugal) (pH 7.4). Biomass was then suspended in the saline solution with a sterile loop and the final solution collected with a pipette tip to a sterile tube. The heavier fragments were allowed to deposit in the bottom for 5–10 min and subsequently the supernatant was transferred to a new sterile tube [26 (link),27 (link)]. Candida strains were maintained on Sabouraud dextrose agar for 48 h at 37 °C [28 (link)]. Bacterial strains were maintained on Tryptic soy agar (TSA) (Liofilchem s.r.l., Roseto D.A., Italy) for 24 h at 37 °C [29 (link)]. Strains were provided by Colección Española de Cultivos Tipo (CECT), Micoteca da Universidade do Minho (MUM), and the American Type Culture Collection (ATCC).

The antimicrobial studies were carried out against eleven microbial reference strains purchased in Frilabo (Maia, Portugal): five Gram-negative bacterial strains (Salmonella Typhimurium ATCC 13311, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Acinetobacter baumannii LMG 1041, and Acinetobacter baumannii LMG 1025); four Gram-positive bacterial strains (Bacillus cereus ATCC 11778, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, and Listeria monocytogenes LMG 16779); and two yeast strains (Candida tropicalis ATCC 750 and Candida albicans ATCC 90028).
Stock cultures were prepared with 20% (v/v) glycerol (Merck, Darmstadt, Germany) and stored at −80 °C. The strains were sub-cultured on an appropriate agar plate 24 h before the antimicrobial studies. Sabouraud dextrose agar (SDA) (Liofilchem, Roseto degli Abruzzi, Italy) was used for the growth of yeasts and brain heart infusion agar (BHI) (Liofilchem, Roseto degli Abruzzi, Italy) was used as culture medium for bacteria [13 (link)].

A total eight clinical isolates, namely, Cn46, Cn47, Cn91, Cn96, Cn118, Cn158, Cn169, and Cn173, were used in this work. The clinical isolates of C. neoformans were obtained from the HIV-positive patients at the Yaoundé Central Hospital and identified by serotyping by multiplex PCR in a previous study [21 (link)]. Sabouraud Dextrose agar (SDA, Liofilchem) and Sabouraud Dextrose broth (SDB, Liofilchem) were used for the activation of yeasts and antimicrobial assays, respectively.

Candida auris NCPF 8971 was stored at −80 ± 2 °C in Sabouraud Dextrose Broth (SDB; Liofilchem, Roseto degli Abruzzi, Italy) with 20% (v/v) glycerol. Before each assay, C. auris was subcultured onto Sabouraud Dextrose agar (SDA; Liofilchem) and incubated aerobically at 37 °C for 18–24 h. SDA plates were prepared from SDB supplemented with 2% (w/v) agar (Liofilchem).

Bacteria and yeast isolates were purchased from ATCC (S. aureus (ATCC 25923), P. aeruginosa (ATCC 89033) and C. albicans (ATCC 90028)) and stored at −80 °C in Luria-Bertani (LB) broth (Liofilchem, Roseto degli Abruzzi, TE, Italy) with 50% glycerol. The experiments were carried out with bacteria cultured in Tryptic Soy Broth (TSB) whereas yeasts in Sabouraud Dextrose Broth (SDB) (Liofilchem) in agitation at 150 rpm (MaxQ 4000, Thermo Scientific, Milan, Italy) or plated on Tryptic Soy Agar (TSA) (Scharlab Italia, Milan, Italy) or Sabouraud Dextrose Agar (SDA) (Liofilchem), respectively, and incubation at 35 °C/37 °C (bacteria) and 25 °C (yeasts).