Supplement of materials and methods
Growth conditions and chemicals: The growth minimal medium (ABTG medium) for biofilm cultivation consisted of 1mM MgCl2, 0.1mM CaCl2, 15.1mM (NH4)2SO4, 33.7mM Na2HPO4, 22mM KH2PO4, 51mM NaCl, 0.01mM FeCl3, 2.5μg/ml Thiamin, and 0.5% Glucose. ABTG medium was refreshed every 24 hours in wells for biofilm cultivation.
Biofilm susceptibility assay: Biofilm assays were performed by a modified Calgary biofilm device method. Isolates were grown overnight in Luria-Bertani (LB) media. After dilution of this culture to ～107cfu/ml, 0.15ml was transferred to all wells except the negative control of a flat-bottom 96-well microtiter plate (catalogue no. 269787; Nalgene Nunc International, Rochester, N.Y.). Bacterial biofilms were formed by immersing the pegs of a modified polystyrene microtiter lid (catalogue no. 445497; Nunc TSP system) into the biofilm growth plate, followed by incubation at 37°C for 1 day, 3 days, and 7 days without shaking, and relative humidity (RH) for biofilm growth plate was above 95% to prevent the evaporation of medium. Crystal violet staining method was employed to check the biofilm formation on pegs for the quality control. Peg lids were rinsed three times in sterile water, placed onto flat-bottom microtiter plates containing antibiotic, twofold dilutions (0-1024μg/ml) in 0.17ml of ABTG medium per well (antibiotic challenge plate), and cultivated for 20h at 37°C. Colistin and Imipenem were tested against all isolates. After antibiotic incubation, peg lids were again rinsed three times in sterile water and placed into antibiotic-free LB in a flat-bottom microtiter plate (biofilm recovery plate) after sonication. To transfer biofilms from pegs to wells, the sonication was performed at 4°C for 2-5 min using Bransonic 220 (80W, 42 KHz, Branson Co., Shelton, Conn. USA). The optical density at 650 nm (OD650) was measured by a microtiter plate reader (Kinetic Microplate Reader, Manufactured by Molecular Devices, Novo Biolab, Denmark) before and after incubation at 37°C for 8-12h. MBIC was defined as the concentrations of the drug that resulted in an OD650 difference at or below 10% of the mean of three positive control well readings. The 10% cutoff represents a 1-log10 difference in growth after 8-12 h of incubation. MBEC was detected after 24h recovery, and defined as the concentrations of drug that resulted in an increase of OD650 <0.05.
Kinetics of antibiotics on biofilm assay
Biofilm cultivation and rinse were described as above for Day1 and Day3. Peg lids were placed into flat-bottom microtiter plates containing different concentrations (0-512μg/ml) of Colistin and Imipenem in 0.17ml of ABTG medium per well (antibiotic challenge plate), and incubated for 0h, 1h, 2h, 3h, 4h, 6h, 8h, 12h, 24h at 37°C. After antibiotic incubation, peg lids were placed into antibiotic-free LB in a biofilm recovery plate after sonication. OD650 was measured by a microtiter plate colorimeter before and after incubation at 37°C for 8-12h.
Killing curves of antibiotics on planktonic cells
Planktonic P. aeruginosa 0.1ml in approximately 106cfu/ml was added into microtiter wells with LB medium. Different concentrations of Colistin or Imipenem (volume 0.1ml) were mixed into different wells, shaken and cultivated at 37°C for hours 0, 1, 2, 4, 8, 12, and 24. Samples (0.1 ml) from wells were cultured overnight on blue plate (Staten Serum Institute, Denmark) and CFU was counted. Killing curves of Colistin and Imipenem on planktonic cells were plotted.
Pharmacokinetics of Colistin and Imipenem in a mouse model of lung biofilm infection
NMRI mice were anesthetized with a mixture of fentanyl and fluanisone (Hypnorm, 10 mg/ml) and Midazolam (Dormicum, 5 mg/ml) in 1:1. P. aeruginosa PAO579 or PAO1 were immobilized in spherical alginate beads. The mice were tracheostomized and 0.04 ml inoculums adjusted to yield approximately 5×108CFU/ml were installed in the lower left lung along the left bronchia using a curved bead-tipped needle. The infected animals were treated intraperitoneally 12h after infection with high dose Colistin (16mg/kg, 6 mice) or Imipenem (64mg/kg, 6 mice). The control groups (6 mice) received equal volumes of 0.15 M NaCl intraperitoneally. The concentrations of Colistin and Imipenem in serum were measured by a biological method with the indicator bacteria Streptococcus sp. EB68 and Bordetella bronchiseptica ATCC4617. Time-concentration curves of Colistin and Imipenem were established. The data of PK running by Kinetica 5.0 (Thermo Fisher Scientific Inc. USA) was plotted against the value of MBIC and MBEC.