Cytoone

Heat-killing (HK) of K. pneumoniae cells was performed in a Thermomixer comfort (Eppendorf) at 95 °C for 30 min in PBS, whereas UV-killing (UVK) was performed in a Stratalinker for 3 cycles at 9999 × 100 µjoules with plate shaking between the cycles. Following HK or UVK, K. pneumoniae cells were added to Aspergillus spp in co-culture in 35 × 10 mm tissue culture dishes (CytoOne, Starlab GmbH, Ahrensburg, Germany) as described above (Strains and growth conditions). Imaging was performed after 48 h incubation. As a control, to check for efficiency of killing, an aliquot (100 µL) of HK and UVK cells was streaked onto LB agar plates and incubated at 37 °C.
Treatment of K. pneumoniae with the antibiotics colistin, kanamycin or tetracycline was performed at the concentrations of 100, 2000 and 10 µg/mL, respectively. Aspergillus spp. and K. pneumoniae were grown alone and in co-culture in YPD medium at 37 °C in 35 × 10 mm tissue culture dishes (CytoOne, Starlab GmbH, Ahrensburg, Germany). Co-cultures of fungi and bacteria were mixed together at time zero, and antibiotics were added to the culture at 6 h. Imaging was performed after 48 h incubation.

Supernatant (SN) of K. pneumoniae growing alone and in co-culture with Aspergillus spp. was obtained after 12 and 24 h growth in biofilm mode in 35 × 10 mm tissue culture dishes (CytoOne, Starlab GmbH, Ahrensburg, Germany). After growth, cells and supernatants were collected into Eppendorf tubes and spun down 2x at maximum speed (25 000 g) at 4 °C (centrifuge 5417 R, Eppendorf, Hamburg, Germany). The SNs corresponding to each growth condition were pooled into 50 mL Falcon tubes, followed by filtration with vacuum filter units (Millipore Express PLUS (PES) 0.22 µm membrane). The freshly collected SN was immediatelly used for growth of Aspergillus spp. Different concentrations of YPD medium were used, including 0.5 volumes SN and 0.5 volumes of 2x YPD or 1x YPD or H₂O. These conditions were intended to mimic exhaustion of nutrients, availability of nutrients and non-exhausted SN, respectively. Imaging of the plates was performed after 48 h incubation. As a control, to check for cell-free supernatants, an aliquot (100 µL) of each SN was distributed on LB agar plates and incubated at 37 °C.

Aspergillus spp. and K. pneumoniae were grown alone and in co-culture in sterile-filtered YPD medium (Formedium, Norfolk, UK) at 37 °C. These were grown as biofilms with static incubation in 35 × 10 mm tissue culture dishes (CytoOne, Starlab GmbH, Ahrensburg, Germany). Co-cultures of fungi and bacteria were mixed together at time zero and incubated for 24 h. An aliquot of these cultures (100 µL) was then streaked on YPD agar plates containing the antibiotic kanamycin 1000 µg/mL to select for fungal growth. Imaging was performed (Canon Macro Lens EF-S 60 mm) after 48 h incubation at 37 °C. As a negative control, to check for absence of bacterial growth, K. pneumoniae strains were streaked alone onto YPD/Kanamycin agar plates. Positive controls were implemented by growing Aspergillus spp. alone in YPD/Kanamycin agar plates to check for unaffected fungal growth in the presence of this antibiotic.