Dnasei

Total RNA was extracted from cells using Tri-Reagent (Sigma-Aldrich) as per the manufacturers’ instructions. SEV RNA was extracted using Trizol LS (Themofisher Scientific Australia Pty. Ltd., Scoresby, VIC, Australia) then purified further using Qiagen RNA Mini kit (Qiagen Pty. Ltd., Chadstone, VIC, Australia). RNA yield was quantified using NanoDrop (Themofisher Scientific Australia Pty. Ltd.) and RNA quality was assessed using either the TapeStation System 2200 or the Bioanalyser (Agilent Technologies, Mulgrave, VIC, Australia). To remove contaminating DNA, 1 μg RNA was treated with 1 μL (2 U) of DNAse I (Themofisher Scientific Australia Pty. Ltd.) in a 20 μL reaction incubated at 37 °C for 30 min and 1 μL DNAse Inactivation reagent added.

Biofilms of K. pneumoniae prepared as described in Section 2.2.1 using a volume of 200 μL were grown over night (~20 h) on 96-well flat-bottomed (Nunc) cell culture plates (polystyrene). Wells were vigorously washed three times with sterile distilled water to remove non-adherent bacteria, filled with 100 μg/mL DNaseI (5-Prime/Termofisher, Hilden, Germany) in PBS, 100 μg/mL proteinase K (Applichem, Darmstadt, Germany) or 1.2 units β-N-acetylglucosaminidase (Sigma, St. Louis, MO, USA) in acetate buffer pH 5.0 and further incubated at 37 °C for 1 h (DNaseI and proteinase K) or 2 h (β-N-acetylglucosaminidase) as previously described [12 (link),13 (link)]. Biofilms incubated with PBS or acetate buffer pH 5.0 were used as controls. After incubation wells were washed once with sterile distilled water and stained for 15 min with 100 μL 1.4% crystal violet at room temperature, washed with distilled water three times to remove excess dye and allowed to dry at room temperature. The crystal violet was dissolved in 100 μL of 95% ethanol (Merck, Darmstadt, Germany) and the optical density at 570 nm was read using a SpectraMax 340PC (Molecular Devices, Sunnyvale, CA, USA).