Col sulfate

In-house BMD was performed according to ISO standard 20776–1 in untreated 96-well polystyrene plates (Greiner bio-one, Frickenhausen, Germany) using cation-adjusted Mueller Hinton II broth (CAMHB, Sigma-Aldrich, Munich, Germany) [15 ]. No additives were included in any part of the testing process (in particular, no polysorbate-80 or other surfactants). COL sulfate was obtained from Sigma-Aldrich (lot no. SLBQ0243V). The final inoculum was adjusted to 2–8 × 10⁵ CFU/ml. Correct inoculum densities were confirmed by obtaining CFU counts of appropriate inoculum dilutions on MH agar plates.

The bioluminescent strain PAO1 carrying luxCDABE gene operon was purchased from Caliper Life Sciences, USA. C1-C22 were clinically isolated from the Chinese PLA General Hospital and identified by the automated Vitek-2 system (bioMérieux, Marcy l’Etoile, France) with a rapid latex agglutination test. C22 was resistant to LEV, AMI, and sulfamethoxazole/trimethoprim. PA ATCC 27853 was used as the quality control strain.
AMK, MER, LEV (Shanghai Macklin Biochemical Co., Ltd., China), and COL sulfate (Sigma, China) were used in our present study. Mueller-Hinton Agar and adjusted Mueller-Hinton Broth (MHA/MHB, Becton, Dickinson and Company) were used to culture bacteria. Pentobarbital (Shanghai Rongchuang Biotechnology Company, China) and isoflurane (Shenzhen RWD Life Science Company, China) were used to anesthetize mice. LIVE/DEAD Bacterial Viability kit (Life Technologies Corporation, Oregon) was used to stain live and dead bacterial cells.

Agar powder (BactoAgar, BD) was added to CAMHB at a concentration of 17 g/L (1.7% agar) [16 (link)]. After autoclaving, the medium was aliquoted, cooled to 50°C and COL sulfate (Sigma-Aldrich) was added at appropriate concentrations to generate working solutions corresponding to a two-fold serial dilution. 100 μl of each aliquot were poured into the appropriate wells of untreated polystyrene 96-well plates. Plates were covered with sterile plastic lids, dried and stored in plastic bags in inverted position at 4 °C. The final inoculum was adjusted to 1 × 10⁴ CFU / well.