Pvp 1

Endolysin XZ.700 was supplied by Micreos (Bilthoven, The Netherlands), and diluted to a solution of 250 mg ml À1 in phosphate buffered saline (PBS, containing NaCl 8 g l À1 , KCl 0.2 g l À1 , Na 2 HPO 4 1 g l À1 , KH 2 PO 4 0.2 g l À1 ) with the addition of 0.1% bovine serum albumin (BSA; Sigma-Aldrich, St Louis, MO, USA). Povidone-iodine (PVP-I; AddedPharma, Oss, The Netherlands) at 0.35% (3.5 mg ml À1 ) (Ruder and Springer 2017) (link), and gentamicin (Sigma-Aldrich, St Louis, MO, USA) at 1000 mg ml À1 , comparable with the tissue concentration when using local gentamicin (Diefenbeck et al. 2006; (link)Mandell et al. 2019) (link), were prepared in PBS from stock solutions containing 10% PVP-I and 50 mg ml À1 of gentamicin, respectively.

All organisms were standardised to a final working concentration of 1 × 10 6 cells/mL in 50% v/v HS (Life Technologies, Paisley, UK) for biofilm development. For viability and biomass assays (described below), 200 µL of single species and triadic species suspensions were added to 96-well flatbottomed microtiter plates (Corning Incorporated, NY, USA). For quantitative polymerase chain reaction (qPCR) and viable cell counting, 500 µL of cultures were added to Thermanox™ coverslips (13 mm diameter, Fisher Scientific) contained within 24 well plates (Corning, NY, USA). Biofilms were incubated at 37°C for 24 h to develop. All procedures were carried out in a class II laminar flow hood. For biofilm development on hydrogels, organisms were standardised to a 1 × 10 6 cells/mL in PBS and added to sections of cellulose matrix (1.25 cm²) (IPS Converters, Oldham, UK). Following initial incubation at 37°C with agitation for 2 h, the matrix was then placed on top of the hydrogel surface and incubated at 37°C for 24 h. Negative controls containing no inoculum were also included. All testing was carried out in triplicate, on three separate occasions. Following biofilm development, cells were washed twice with PBS to remove any non-adherent cells before treatment with 10% w/v PVP-I (Sigma) or 0.05% v/v CHX (Sigma) for a further 24 h at 37°C. Untreated controls were also included.