Sabouraud broth

The antibacterial activity was examined by the agar disc diffusion method. Bacterial strains of S. maltophilia and B. subtilis were incubated in the Mueller Hinton broth (MHB, Oxoid, Basingstoke, UK) at 37 °C for 24 h. P. expansum were incubated in the Sabouraud broth (SB, Oxoid, Basingstoke, UK) at 25 °C for 48 h. S. maltophilia, B. subtilis and P. expansum isolates were identified by MALDI-TOF MS Biotyper (Brucker, Daltonic, Germany) with high scores, 2.345, 2.234 and 2.425, respectively. Microbial suspensions in saline water of 0.5 McFarland turbidity standards (densitometer, Erba Lachema s.r.o., Brno, Czech Republic) were streaked onto Mueller-Hinton agar (MHA) and Sabouraud agar with a sterile swab. A sterile filter disc (diameter 6 mm, Whatman paper N. 1) was impregnated with EOs (10 µL/disc). The inoculated MHAs were placed at 4 °C for 2 h and then incubated at 37 °C, resp. 25 °C for 18–24 h, resp. 48 h. The antimicrobial activity was evaluated by measuring the zones of growth inhibition. Gentamicin (10 µg, Oxoid, Basingstoke, UK) was used as the positive control. The experiment was performed in triplicate.

The bioluminescent bacterial strains, P. aeruginosa (strain P1242) and S. aureus (Xen29) and the fluorescent fungal strain (GFP-tagged strain derived from C. albicans SC5314) were used. According to previously detailed protocols [11 (link)], the viable bacterial or fungal cells constitutively emitted bioluminescent or fluorescent signals that could be recorded and quantified by a Fluoroskan reader (Thermo Fischer Scientific, Waltham, MA, USA). Such values, expressed as Relative Luminescence Units (RLU for bacterial cells) or Relative Fluorescence Units (RFU for fungal cells), allowed us to directly measure the amounts of viable microorganisms present in the control and experimental groups.
Operationally, in line with other studies [16 (link)], bacterial and fungal cells from −80 °C glycerol stocks were initially seeded onto Tryptic Soy Agar (TSA) or Sabouraud Dextrose Agar (SAB) (OXOID, Milan, Italy) plates, respectively, and incubated overnight at 37 °C. Then, isolated colonies were collected, added to 10 mL of Tryptic Soy Broth (TSB) or Sabouraud broth (OXOID, Milan, Italy) and allowed to grow overnight at 37 °C under gentle shaking, prior to being used for biofilm production.

Microbial strains belonged to the microbial collection of the Department of Agricultural and Food Sciences, University of Bologna (Italy) [11 (link),14 (link),15 ]. Lactobacillus plantarum 98a, Lb. sanfrancisciensis Bb12 and Saccharomyces cerevisiae LBS were obtained from 30% (v/v) glycerol stocks stored at -80 °C. Bacteria were propagated in de Man–Rogosa–Sharpe (MRS) (Oxoid, Thermo Scientific, Waltham, MA, USA) broth at 37 °C for at least 48 h and yeasts in Sabouraud broth (Oxoid, Thermo Scientific, USA) at 30 °C for 24 h.

Candida albicans strains used in this study included SC5314 (wild type), CAI4 (wild type), MBY38 [tetO-UME6 (Carlisle et al., 2009 (link))], efg1Δ/cphΔ1 (Lo et al., 1997 (link)), nrg1Δ (Murad et al., 2001 (link)), hgc1Δ (Zheng and Wang, 2004 (link)) and tup1Δ (Braun and Johnson, 1997 (link)). Strains were routinely grown and maintained on YPD agar (Sigma-Aldrich). For the MBY38 strain, plates contained 40 μg ml⁻¹ of doxycycline. For inoculum preparation, a single colony was grown in Sabouraud broth (Oxoid) at 30°C for 24 h with shaking. Cells were washed twice in sterile phosphate-buffered saline (PBS) and counted using a haemocytometer. Inoculums of the filamentous strains, nrg1Δ and tup1Δ, were standardized to 1 × 10⁷ CFU ml⁻¹ of SC5314 by quantifying protein concentration (Meyers et al., 1998 (link)).