A panel of 15 fungal species comprising Candida albicans ATCC MYA-2876 and 10231, Candida glabrata ATCC 2001, Candida auris NCPF 8978 and 8973, Candida tropicalis BC064, Candida parapsilosis NCPF 8334, Candida haemolunii DSM 70624, Malassezia furfur NCPF 3349, Rhodotorula spp., Trichosporon spp., Aspergillus brasiliensis ATCC 16404, Aspergillus fumigatus NCPF 7367, Scedosporium spp., and Rhizopus spp. was assembled for this study. Strains were maintained on Sabouraud dextrose (SAB) agar (Oxoid, Hampshire, United Kingdom) and refrigerated at 4°C prior to proliferation in yeast-peptone-dextrose (YPD; Sigma-Aldrich, Dorset, United Kingdom) in a 200-rpm shaking incubator overnight at 30°C for Candida, Malassezia, Trichosporon, and Rhodotorula species. Spores of the pore-forming fungi Aspergillus, Rhizopus, and Scedosporium were harvested using a previously described method for storage at 4°C (63 (link)). Spores and yeast cells were pelleted through centrifugation at 3,000 × g and washed twice with phosphate-buffered saline (PBS). Cells and spores were then counted using a Neubauer hemocytometer and standardized to a working concentration for further experimentation.
Sabouraud dextrose sab agar
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, Italy
Sabouraud dextrose (SAB) agar is a microbiological culture medium used for the isolation and cultivation of fungi, particularly yeasts and molds. It provides a suitable environment for the growth of these microorganisms by supplying dextrose as a carbon source and maintaining a slightly acidic pH range.
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Lab products found in correlation
Yeast peptone dextrose ypd, by Merck Group (3 mentions)
Rpmi 1640, by Merck Group (2 mentions)
Potato dextrose agar (pda), by Thermo Fisher Scientific (1 mentions)
Microbank beads, by Pro-Lab Diagnostics (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Trypticase soy broth (tsb), by Thermo Fisher Scientific (1 mentions)
Mrs broth, by Thermo Fisher Scientific (1 mentions)
Fluoroskan reader, by Thermo Fisher Scientific (1 mentions)
Sabouraud broth, by Thermo Fisher Scientific (1 mentions)
8 protocols using sabouraud dextrose sab agar
1
Fungal Species Panel for Antimicrobial Assays
2
Dermatophyte Fungal Isolate Sourcing and Culturing
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A complete list of fungal isolates used in these experiments can be found in Table S1. Dermatophytes were purchased from the National Collection of Pathogenic Fungi (Public Health England, UK), the American Type Culture Collection (LGC Standards, UK), and the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmBH (DSMZ, Germany). Clinical isolates of dermatophytes were obtained from Michel Monod (Centre Hospitalier Universitaire Vaudois, Switzerland) and Boni Elewski (University of Alabama at Birmingham). Dermatophyte cultures were grown on Sabouraud dextrose (SAB) agar (Oxoid, Ltd., UK), for up to 14 days at 30°C. Fungi were stored at –85°C with 0.5% (vol/vol) dimethyl sulfoxide as cryoprotectant. For experimental purposes, dermatophytes were cultured at 30°C for up to 14 days on PDA (Oxoid, Ltd).
3
In vitro biofilm assessment of C. auris
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For in vitro biofilm biomass assessment, a pool of aggregating (Agg; n = 12) and single-celled, nonaggregative (non-Agg; n = 14) C. auris clinical isolates (gift from Andrew Borman and Elizabeth Johnson, Public Health England, UK). The isolates used and the clades they belong to are shown in Table 1 . All C. auris isolates were stored in Microbank beads (Pro-Lab Diagnostics, UK) prior to use. Each isolate was grown on Sabouraud dextrose (SAB) agar (Oxoid, UK) at 30°C for 24 to 48 h and then stored at 4°C prior to propagation in yeast-peptone-dextrose (YPD; Sigma-Aldrich, UK) medium overnight (16 h) at 30°C, gently shaking at 200 rpm. The cells were pelleted by centrifugation (3,000 × g) and then washed two times in phosphate-buffered saline (PBS). The cells were then standardized to the desired concentration after counting using a hemocytometer and then resuspended in selected media for each assay, as described in this article.
C. auris isolate phenotypes were determined visually by suspending one colony in 1 ml of PBS (Sigma-Aldrich, UK). Isolates were termed “aggregators” if the added colony did not disperse upon mixing in PBS. For RNA sequencing and transcriptional analysis of C. auris biofilms and coculture systems, one Agg isolate (NCPF 8978) and one non-Agg isolate (NCPF 8973) were used.
4
Candida auris Isolation and Preparation
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Throughout this study four Candida auris (Ca) isolates obtained from various clinical sites [7] , (NCPF 8971, NCPF 8973, NCPF 8977, NCPF 8978) were used, as previously described [3] . All isolates were identified by ribosomal DNA (rDNA) gene sequencing or matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) [7] . Candida glabrata (Cg) ATCC 2001 and Candida albicans (Ca) ATCC 10231 was used as reference strains. All strains were stored and maintained on Sabouraud dextrose (SAB) agar (Oxoid, Hampshire, UK) prior to propagation in yeast peptone dextrose (YPD) (Sigma-Aldrich, Dorset, UK) medium overnight at 30°C. Cells were prepared according to a modified version of the British Standards for chemical disinfectants and antiseptics [8] . Briefly, cells were washed by centrifugation in phosphate buffered saline ([PBS] Sigma-Aldrich, Dorset, UK), and standardised to 1 x 10 7 cells/mL in sterile water containing 5% foetal bovine serum to simulate organic material.
5
Antimicrobial Susceptibility Profiling
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Anonymized clinical Staphylococcus aureus PECHA 10, Pseudomonas aeruginosa PECHA 4, and Candida albicans X3 strains (Di Giulio et al., 2018 (link), 2020 (link)) were used for this study. The strains were isolated from chronic wounds of patients that gave their informed consent for the study. The study was approved by the Inter Institutional Ethic Committee of University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy (ID n. richycnvw). The strains were characterized for their susceptibility to antibiotics, and in particular, S. aureus PECHA 10 and P. aeruginosa PECHA 4 were resistant strains (Supplementary Table 1 ). All the methods were performed in accordance with the relevant guidelines and regulations. For the experiments, bacteria were cultured in Trypticase Soy Broth (TSB, Oxoid, Milan, Italy) and incubated at 37°C overnight in aerobic condition and then refreshed for 2 h at 37°C in an orbital shaker. Then the broth cultures were standardized to optical density at 600 nm (OD600) = 0.125. The broth culture of Candida albicans, grown on Sabouraud dextrose agar (SAB, Oxoid, Milan, Italy) was prepared in RPMI 1640 (Sigma-Aldrich, Milan, Italy) plus 2% glucose and standardized to OD600 = 0.15.
6
Probiotic Strain Preparation for Adhesion
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The probiotic strains used for this study are reported in Table 1 . The bacterial strains were stored at −20 °C in MRS broth (Oxoid, Milan, Italy), whereas the yeasts were stocked on Sabouraud dextrose agar (SAB; Oxoid, Milan, Italy) slants at 4 °C. Before each assay, the bacterial and yeast strains were grown in their optimal media (OM), at their optimal conditions (see Table 1 ), until late exponential phase was attained. Cells cultures were successively harvested by centrifugation for 10 min at 4,500 rpm (4 °C) and the pellets were washed twice with sterile isotonic solution temperate at 4 °C and finally resuspended in physiological solution (0.9% NaCl) at a cell concentration of 1 × 108 CFU mL−1. These cell suspensions were diluted in order to make a cell concentration of 103 CFU mL−1 (working culture) for adhesion experiments. To guarantee reproducibility in the inocula preparation, the cell counts were standardized through the direct plate count method (Harrigan, 1998 ).
7
Quantifying Microbial Viability via Bioluminescence and Fluorescence
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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.
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.
8
Antifungal Resistance Profile of Candida albicans S5
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A clinical strain of Candida albicans S5, isolated from the oral cavity of healthy individuals and collected at the Department of Medical, Oral, and Biotechnological Sciences, was used for this study (reference number: BONEISTO N. 22-10.07.2021, University G. d’Annunzio Chieti-Pescara, 10 July 2021, approved by the Inter Institutional Ethic Committee of University “G. d’Annunzio” Chieti-Pescara, Chieti, Italy). The resistance profile of C. albicans S5 towards antifungal drugs commonly used in therapy was analyzed. The strain for the experiments was grown on Sabouraud dextrose agar (SAB, Oxoid, Milan, Italy), cultured in RPMI 1640 (Sigma-Aldrich, Milan, Italy) plus 2% glucose, and standardized to obtain a suspension containing ≈107 CFU/mL, optical density/OD600 = 0.15, as performed using a spectrophotometer (Eppendorf, Milan, Italy).
For the biofilm preparation, 200 µL of standardized C. albicans S5 suspension was incubated on 96-well flat-bottomed microtiter plates at 37 °C for 24 h [12 (link)]. Subsequently, the wells were rinsed three times with sterile PBS to remove the planktonic cells, and then treated with ALAD-aPDT as described.
For the biofilm preparation, 200 µL of standardized C. albicans S5 suspension was incubated on 96-well flat-bottomed microtiter plates at 37 °C for 24 h [12 (link)]. Subsequently, the wells were rinsed three times with sterile PBS to remove the planktonic cells, and then treated with ALAD-aPDT as described.
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