Masterflex silicone tubing

For biofilm antibiotic susceptibility and RNA extraction, biofilms were grown in a continuous-flow reactor system with size 13 (1 m in length) Masterflex silicone tubing (Cole Parmer) at a flow rate of 0.1 ml/min, as previously described using 20-fold-diluted LB medium (3 (link)– (link)5 ). For protein extraction, biofilms were grown in a continuous-flow reactor system with size 14 (1 m in length) Masterflex silicone tubing (Cole Parmer) at a flow rate of 0.2 ml/min using 20-fold-diluted LB medium. Carbenicillin at 10 μg/ml was added to the growth medium for plasmid maintenance. To visualize and quantify biofilm formation, biofilms were grown in 24-well plates in 5-fold-diluted LB medium containing 10 μg/ml carbenicillin, with the growth medium being exchanged every 12 h as previously described (23 (link)). Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Inc., Wetzlar, Germany) and the LIVE/DEAD BacLight bacterial viability kit (Life Technologies, Inc.). Quantitative analysis of the confocal laser scanning microscope images of 24-well plate-grown biofilms was performed using COMSTAT (54 (link)).

For biofilm antibiotic susceptibility, biofilm MBC assays, protein extraction, and RNA extraction, biofilms were grown in a once-through continuous flow reactor system with size 13 (inner surface area, 25 cm²) Masterflex silicone tubing (Cole Parmer) for 3 days at a flow rate of 0.1 ml/min, as previously described using 20-fold-diluted LB medium (3 (link)– (link)5 (link)). To maintain plasmids, carbenicillin at 10 µg/ml was added to the growth medium. For visualization of the biofilm architecture, biofilms were grown in 24-well plates in 5-fold-diluted LB medium containing 10 µg/ml carbenicillin, with the growth medium being exchanged every 12 h. Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 confocal microscope (Leica Microsystems, Inc., Wetzlar, Germany) and the LIVE/DEAD BacLight bacterial viability kit (Life Technologies, Inc.). Quantitative analysis of the confocal laser scanning microscope images of 24-well plate-grown biofilms was performed using COMSTAT (7 (link)). For all biofilm growth conditions, wild-type strain PAO1 and the ΔsagS mutant strain harboring empty plasmids were used as vector controls.