Polycarbonate membranes

Lyophilised 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was dissolved in chloroform, with or without head group-functionalized lipid (i.e. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl) (referred to here as BC-PE) or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] (termed PDP-PE)) (Avanti Polar Lipids, USA), within a glass tube. Chloroform was removed under a continuous nitrogen stream to produce a thin lipid film. Tubes were placed under vacuum for a minimum of 2 h to ensure all chloroform had been removed. Lipid films were rehydrated in 20 mM HEPES, 150 mM NaCl, 2 mM CaCl₂, pH 7.4 at 1 mg ml⁻¹. Lipid solutions were extruded through a 50 nm pore size filter composed of polycarbonate membranes (Whatman®, GE Healthcare Life Sciences), using a mini-extruder set (Avanti Polar-Lipids, USA). Dynamic Light Scattering (DLS) using a Zetasizer nano-S instrument (Malvern Instruments Ltd, UK) was used to check the size of the unilamellar vesicles. Only vesicles producing single peaks with a z-average of approximately 100 nm and below were used.

Overnight grown S. pneumoniae R6st cells were diluted to approximately 1 × 10⁶ CFU/mL, and biofilms formed for 24, and 48 h on polycarbonate membranes (0.1 μm, 25 mm, UV-sterilised on both sides, Whatman, GE Healthcare Life Sciences, PA, USA) as previously described [29 (link)]. Membranes were placed with the shiny side up onto TSA_sb plates and inoculated with 50 μL of culture, and incubated upright (37 °C, 5 % CO₂), being transferred to a fresh plate every 24 h. For colony biofilm treatment with MSlys, membranes were transferred to 6-well plates, and 50 μL of MSlys (final concentration of 4 μM) or PBS (negative control) were applied on the whole surface and incubated for 30 min, 1 h or 2 h.