50 ml falcon tube

Subsoil for DNA extraction optimization was collected on July 20, 2019 from a Phaeozem soil at a conventional agricultural field near Dornburg, Thuringia, Germany (Table S1). A soil core (Ø 10 cm) was obtained using a steel cylinder, with a hardened steel cutting head driven into the soil by an electric caulking hammer (Makita HM1400, Makita, Fischamend, Austria). Subsoil was collected from 60 cm depth and the outer few millimetres (approximately 5 mm) of the soil core were removed to avoid a transfer of the topsoil into the subsoil material (carryover) during the sampling procedure. The subsoil was homogenized in a sterile polyethylene bag and approximately 50 g of fresh soil was transferred into a sterile 50-mL Falcon tube (SARSTEDT, Nümbrecht, Germany) and frozen at −20 °C in the field. Upon arrival at the laboratory, the subsoil sample was freeze-dried for 72 h and subsequently homogenized using a swing mill (Retsch MM400, Retsch, Haan, Germany) at 25 Hz for 1 min. Homogenized soil was stored air-tight in the dark at room temperature.

One discrete
colony of each strain was selected, transferred with a sterile plastic
loop (VWR International), and inoculated into a 50 mL Falcon tube
(Sarstedt, Nümbrecht, Germany) with 10 mL of nutrient-rich
general media: tryptic soy broth (TSB), BD Difco, USA) for S. aureus and LB [low NaCl] (10 g/L tryptone, 5 g/L
yeast extract, and 0.5 g/L NaCl) (BD Difco, USA) for P. mirabilis. The pre-cultures were grown overnight
in a rotating incubator (New Brunswick Scientific, Innova 40/40R Incubator
Shakers, Eppendorf AG, Hamburg, Germany) at 37 °C and 200 rpm
shaking for 18 ± 1 h. After overnight incubation and prior to
starting the bacterial growth assays, the pre-cultures were washed
thoroughly to remove the rich nutrient broth and secreted metabolites.
The pre-cultures were centrifuged (3220g, 10 min,
20 °C, 5810R table centrifuge, Eppendorf AG, Hamburg, Germany)
and the cell pellets were washed twice in 20 mL of 0.9% sterile NaCl
solution (Merck Millipore, Darmstadt, Germany) and finally re-suspended
by vortexing in 10 mL of 0.9% sterile NaCl solution. The optical density
(OD) of the washed pre-cultures was measured at 620 nm [ultraviolet
(UV)/visible spectrophotometer Ultrospec 2100 pro, GE Healthcare,
Little Chalfont, UK] and the adequate inoculum volume to obtain a
starting OD of 0.1 in 200 μL was calculated.

From the used towels, a 4 × 4 cm (16 cm²) piece was cut out and placed in a 50 mL falcon tube (Sarstedt AG & Co. KG, Nümbrecht, Germany) filled with 20 mL of sterile 0.9% NaCl. The tubes were placed on a roll shaker (MaxQ*, Thermo Fisher Scientific Inc., Waltham, Massachusetts, USA) for 20 min followed by vigorous vortexing for 1 min and centrifugation for 20 min/8 °C/4696 g.
After centrifugation, the supernatant was discarded and the remaining pellet was resuspended in 700 µL of sterile 0.9% NaCl, which was used both for the determination of the bacterial count and for DNA extraction using the “Fast-DNA SPIN Kit for Soil” (MP Biomedicals GmbH, Eschwege, Germany).
For the determination of the bacterial counts, a decimal dilution series was prepared and 100 µL of each sample was spread on Tryptic Soy Agar to detect aerobic mesophilic bacteria (TSA, Merck KGaA, Darmstadt, Germany), Malt Extract Agar to detect yeasts and moulds (MEA, Merck KGaA, Darmstadt, Germany), MacConkey to detect Gram-negative bacteria (Merck KGaA, Darmstadt, Germany), Mannitol Salt Agar to detect Staphylococcus spp. (MSA, Xebios Diagnostics GmbH, Düsseldorf, Germany), and Cetrimide Agar to detect Pseudomonas spp. (Xebios Diagnostic GmbH, Düsseldorf, Germany)). Incubation was performed at 30 (MEA, MSA, and Cetrimide) or 37 °C (TSA, MacConkey).