Achemidoc touch imaging system

PIA detection was performed as described by Cerca et al. (2006). Biofilm samples were grown and
collected following the PBS washing step as described in Sect. 2.3. Samples
were resuspended in 50  $\mathrm{\mu }\mathrm{L}$ of 0.5 M EDTA (pH 8.0) and incubated for 5 min at 100  ${}^{\circ }$ C. Next, samples were centrifuged for 10 min
at $\mathrm{5000}\times$   $g$ . An amount of 40  $\mathrm{\mu }\mathrm{L}$ of the supernatant was collected and incubated
with 10  $\mathrm{\mu }\mathrm{L}$ proteinase K (20 mg/mL) for 30 min at 37  ${}^{\circ }$ C.
Samples were then mixed with 10  $\mathrm{\mu }\mathrm{L}$ of 20 mM Tris pH 7.4, 150 mM NaCl,
and 0.1 % bromophenol blue. An amount of 3  $\mathrm{\mu }\mathrm{L}$ of the preparation and
dilutions was spotted onto a nitrocellulose filter, blocked with 3 % BSA
in TBS 0.1 % Tween (TBST) for 2 h at room temperature. Then samples
were incubated with PIA antibody (L3892; Sigma) overnight at 4  ${}^{\circ }$ C.
The filter was washed five times in TBST. PIA was detected by ECL
chemiluminescent detection reagent (Thermo Scientific) according to the manufacturer's instructions. Images were developed and quantified using a
ChemiDoc Touch imaging system (Bio-Rad). In an independent biofilm assay,
bacteria were treated with 10  $\mathrm{\mu }\mathrm{M}$ sodium metaperiodate or PBS 2 h
prior to experimental endpoints. Crystal violet was used to stain the
biofilm, then it was dissolved in 30 % acetic acid. The absorbance of the biofilm
was measured at 600 nm. All results are expressed as a mean value  $\pm$  standard deviation.