In addition, the entire contents of the resuspended biofilm were plated onto LB10 agar supplemented with 300 μg mL−1 of rifampicin (Sigma Aldrich) to quantify the number of spontaneous rifampicin-resistant mutants. The plates were incubated for 2 days at 37 °C after which time CFUs were enumerated. The mutation frequency was calculated as the number of spontaneous rifampicin-resistant mutants divided by the total viable population. The ability of each variant to utilise different see more substrates as carbon sources was determined using the commercially available

BIOLOG GN2 plates (Biolog, CA) according to the manufacturer’s instructions (minor modifications as below). Each plate contains 95 different carbon sources, each conjugated to a tetrazolium

dye. The ability to utilise a specific substrate results in dye cleavage and the formation of a purple hue in the wells. In brief, bacterial cultures were grown overnight in 10 mL of M9 medium (supplemented check details with 5.5 mM glucose) at 37 °C with shaking. Following centrifugation (4580 g) and washing (twice with 10 mL PBS), bacteria were resuspended in 20 mL of GN2 inoculating fluid (Biolog). The BIOLOG GN2 plates were then inoculated with 150 μL of the resuspended bacteria and incubated at 37 °C. The OD600 nm was taken at 0, 4, 8 and 24 h (Wallac Victor2 plate reader; Perkin Elmer) to monitor the growth of cells within each well. A dye release profile corresponding to the amount and types of carbon sources metabolised was generated for the 24-h time point. The quantification of attachment from and batch biofilm formation was conducted on both polystyrene- (hydrophobic) (Sarstedt Inc) and tissue culture–treated (hydrophilic) (Costar, Corning Inc) 96-well microtitre plates using an assay similar to that described previously (O’Toole & Kolter, 1998; Pratt & Kolter, 1998; Koh et al., 2007). Briefly, for attachment, 100-μL aliquots of overnight cultures in LB10 were added into the wells, while for biofilm formation, overnight cultures were diluted 1 : 100 in LB10 broth. Subsequently, 100-μL aliquots of the diluted cultures were added into the wells,

and the plates were incubated without agitation at 37 °C for 2 h for attachment and/or 24 h with shaking for biofilm formation. After incubation, the cell density of each well was determined (OD600 nm), the cell suspensions were removed, the wells were washed twice with PBS, 100 μL of filtered 1% (w/v) crystal violet (CV) solution was added into each well, and the plates were incubated at room temperature for 20 min. The CV solution was removed, and the wells were washed three times with PBS followed by the addition of 100 μL of HPLC-grade absolute ethanol (Univar) to extract the CV for quantification at OD490 nm. For the attachment assay, the CV reading was normalised using the cell density reading (OD490 nm/OD600 nm).