Sodium chloride (nacl)

The starting calcium phosphate powder (β-TCP, 96%, Product No. 21218) was supplied by Sigma-Aldrich (Poznań, Poland) as a product of Fluka Chemie GmbH, Buchs, Switzerland, and the 30% solution of H₂O₂ (Catalog No. BA5193111) was supplied by Avantor Performance Materials Poland S.A., Gliwice, Poland. In this research, we used the poly (l-lactide) Resomer L210s (PLLA) supplied by Evonik, Germany; 1,4-dioxane (CAS:123-91-1) was supplied by Eurochem BGD (Tarnów, Poland) and sodium chloride (CAS: 7647-14-5) for salt leaching experiments was purchased from Stanlab (Lublin, Poland). l-lysine for HAP modification, (CAS: 56-87-1) was purchased from Sigma-Aldrich (Poznań, Poland).

The 0.5 McFarland (MF) density (established using densitometer Densitomat II, BioMerieux, Warsaw, Poland) of the bacteria suspension in specific medium (TSB, TSB+G, DMEM) was prepared and next diluted to 1 × 10⁵ CFU/mL. A total of 100 μL of the suspension was added to the six wells of a 96-well microtiter plate (VWR, Radnor, PA, USA) and incubated for 24 h at 37 °C. Subsequently, the non-adhered cells were removed, and the plate was dried for 10 min at 37 °C. Next, 100 μL of 20% (v/v) water solution of crystal violet (Aqua-med, Lodz, Poland) was added, and the mixture was incubated for 10 min at room temperature. After incubation, the solution was removed, the biofilm was gently washed twice with 100 μL of 0.9% NaCl (Stanlab, Lublin, Poland), and dried for the next 10 min. A total of 100 μL of 30% water solution of acetic acid (v/v) (Chempur, Piekary Slaskie, Poland) was then introduced to the plate; the whole setting was subjected to mechanical shaking at 450 rpm for 30 min (Schuttler MTS-4, IKA, Königswinter, Germany). After shaking, the solution was transferred to a fresh plate, and the absorbance of extracted CV was measured at 550 nm wavelength using a MultiScan Go Spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA). The level of biofilm biomass was performed for each strain in six replications in three independent experiments.

The 0.5 McFarland density of the bacteria suspension in TSB medium was prepared, then diluted to 1 × 10⁵ CFU/mL. A total of 1 mL of the suspension was added to the well of a 24-well microtiter plate (Wuxi Nest Biotechnology, Wuxi, China) and incubated for 24 h at 37 °C. Subsequently, the non-adhered cells were removed, and the plate was dried for 10 min at 37 °C. Next, 1 mL of 20% (v/v) water solution of crystal violet (Aqua-med, Lodz, Poland) was added, and the mixture was incubated for 10 min at room temperature. After incubation, the solution was removed, the biofilm was gently washed twice with 100 μL of 0.9% NaCl (Stanlab, Lublin, Poland) and dried for the next 10 min. Next, the image of the dyed biofilm was captured photographically.

To determine the appropriate concentration of gentamicin needed to saturate BC-coated meshes, the amount of BC on meshes had to be specified. For this purpose, pieces of meshes were aseptically weighed (Pioneer PA 114CM/1, OHAUS, Parsippany, NJ, USA) before coating with BC and after coating, purification and sterilization processes. Samples were prepared with concentrations of MIC (determined during current research) and 4.0 mg/mL of gentamicin (Oxoid, Thermo Fisher Scientific, Hampshire, UK) in BC-coated and uncoated meshes. Samples were placed in 24-well plates (VWR, Radnor, PA, USA), and 0.5 mL of gentamicin solutions was added. Incubation lasted for 24 h at 4 °C. As a negative control (no antimicrobial effect), samples were saturated with 0.9% of NaCl (Stanlab, Lublin, Poland). The release profile of gentamicin (4 mg/mL) from uncoated and coated meshes was determined analogically to the procedures performed in our earlier publication [57 (link)].

Hyphae of P. infestans were frozen in liquid nitrogen and stored at 80 °C until use. For DNA extraction hyphae (0.150 g) were ground to a fine powder and then homogenized in buffer containing 200 mM Tris-HCl (pH 7.5) (Bio-Rad; Hercules, CA, USA), 250 mM NaCl (Stanlab; Lublin, Poland), 25 mM EDTA (BioShop; Mainway, Burlington, ON, Canada), 10% SDS (Sigma; Saint-Louis, MO, USA), and RNase A (ThermoFisher; Waltham, MA, USA) was added to each sample. After incubation (30 min at RT with mixing) the phenol-chloroform-isopropanol mixture (1:2:1) (BioShop; Mainway, Burlington, ON, Canada) was added and samples were mixed. After centrifugation (10,000 g for 12 min at 4 °C) the upper layer was collected, mixed with chloroform, and centrifuged (10,000 g for 12 min at 4 °C). Then the upper layer was mixed with isopropanol, incubated for 10 min at RT, and centrifuged (10,000 g for 12 min at 4 °C). The supernatant was removed, the precipitate was air dried, and dissolved in H₂O_DEPC.

Superoxide dismutase was assayed by measuring SOD ability to inhibit the photochemical reduction of NBT using the method of Beauchamp and Fridovich [59 (link)]. Fresh hyphae (0.05 g) were homogenized in 0.05 M sodium-phosphate buffer (pH 7.0) (Chempur; Piekary Śląskie, Poland), containing 1 mM EDTA (BioShop; Mainway, Burlington, ON, Canada), 1% PVPP (Sigma; Saint-Louis, MO, USA), and 0.01 M NaCl (Stanlab; Lublin, Poland). After centrifugation (15,000 g for 30 min at 4 °C) the supernatant was collected and used for assays. The assay mixture contained 0.05 M sodium phosphate buffer (pH 7.8) (Chempur; Piekary Śląskie, Poland), 13 mM methionine (Sigma; Saint-Louis, MO, USA), 75 μM NBT (SERVA; Heidelberg, Germany), enzymatic extract, and 2 μM of riboflavin (SERVA; Heidelberg, Germany). The reaction was initiated by UV radiation (15 W) and was run for 15 min. The absorbance was measured at a wavelength of 560 nm. The amount of the enzyme that caused inhibition of the NTB reduction reaction by 50% was assumed as a unit of SOD activity (U × mg⁻¹ protein). To determine SOD isoenzymes a samples containing 50 μg of protein were separated in a 10% nondenaturing acrylamide gel and visualized using the method of Beauchamp and Fridovich [59 (link)].