Tobramycin

P. aeruginosa was grown in chamber slides as previously described^{63 (link)}. Briefly, clinical isolates of P. aeruginosa were grown overnight in 3 mL of lysogeny broth (Lennox formulation-LB) with shaking overnight. 40 μL of overnight culture was diluted into 4 mL of LB. This was diluted 1/10 and 220 µL was used to seed the wells of an 8-chambered cover-glass slide (Nunc Lab-tek II, VWR, Mississauga, Ontario, Canada). After 6 hours of attachment, media were removed and replaced with fresh media. Biofilms were allowed to grow for a further 24–48 hours, replacing media every 12 hours until the conclusion of the experiment. Pre-formed biofilms in chamber slides were also exposed to varying concentrations of cationic peptide, tobramycin (Sigma-Aldrich, Oakville, Ontario, Canada) or a combination of both as described. 24-hour biofilms were grown as described above prior to addition of various antibiotics for the indicated time periods.

HeLa cells were cultured at 37 °C and 5% CO₂ in low glucose Dulbecco’s minimal essential medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% glutamine, 100 units/mL penicillin and 100 µg/mL streptomycin. 1 × 10⁵ cells were seeded per well in a 12-well plate and transfected using Effectene transfection reagent (Qiagen) according to the manufacturer’s instructions. Transfection reagent was removed six hours after transfection. The cells were treated with the respective TRIDs and incubated for another 18 h. Amikacin, erythromycin, josamycin, paromomycin, tobramycin and tylosin were obtained from Sigma-Aldrich, gentamicin and G418 from Carl Roth. Stock solutions were made in water, except for erythromycin (70% ethanol).

MICs were determined in triplicate according to the EUCAST broth microdilution method [47 ] in U-bottom 96-well microtiter plates using LB and ASM. Nalidixic acid, amikacin, aztreonam, ciprofloxacin, minocycline, piperacillin and tobramycin were obtained from Sigma-Aldrich (Merck Millipore, Burlington, MA, USA). Levofloxacin and sparfloxacin were obtained from Honeywell Fluka™ (Charlotte, NC, USA) and Meropenem from AstraZeneca (Cambridge, UK). The MIC was determined via the resazurin method [48 (link)].

A clinical isolate of A. baumannii (strain ATCC 17978; “Ab17978”; originally isolated from an infant) was used in the present study. The strain was resistant to at least three different categories of antibiotics, including beta lactams. Luria–Bertani (LB, Becton Dickinson and Company, Sparks, MD, USA) agar and broth was used for bacterial culture. Ampicillin, tobramycin, chloramphenicol, erythromycin, ciprofloxacin, maltodextrin, sucrose, 3350 Da polyethylene glycol (PEG), and 400 Da PEG were procured from Sigma-Aldrich (St. Louis, MO, USA). tobramycin (Tob), chloramphenicol (Chl), erythromycin (Ery) and ciprofloxacin (Cip) were selected for sequential treatment because they represent four different classes of antibiotics with different mass (565, 323, 733, and 331 Da, respectively) and Log P characteristics (−6.2, 1.1, 2.7, and −1.1 units, respectively). Log P is the partial coefficient between n-octanol and water, and larger negative values represent more hydrophilic compounds. Log P and mass values used in this study were obtained from PubChem Compound https://www.ncbi.nlm.nih.gov/pccompound/.

hNE (R1162X/R1162X) cells were seeded in T75 flasks coated with collagen IV (Sigma) or Purecol (Advanced Biomatrix) in 12 ml pre-warmed complete PneumaCult ALI Ex⁺ medium (StemCell kit) at 37 °C in 5% CO₂. To 500 ml medium the following supplements were added: 0.5 ml hydrocortisone (StemCell), 10 ml 50X Ex⁺ supplement (StemCell kit), and for some preparations 2 ml of amphotericin B (12.5 µg ml⁻¹; Sigma), 500 µl ceftazidime (100 mg ml⁻¹; Sigma), 500 µl vancomycin (100 mg ml⁻¹; Sigma), and 500 µl tobramycin (100 mg ml⁻¹; Sigma). Cells were expanded for 3–5 days, until they reached 70–80% confluency.
Cells were then detached by 0.05% trypsin-EDTA (Pan Biotech) or enzymatic (StemCell) treatment and seeded onto 12- or 24-ALI Transwells (0.4-µm pore polyethylene terephtalate membrane inserts, Corning) coated with collagen IV at a confluency of 1.5 × 10⁵ to 2 × 10⁵ per well, and Complete Ex⁺ medium (without antibiotics) was added as following, 0.5–0.6 ml (basolaterally) and ~0.5 ml (apically). Cells were grown for 3–4 days at 37 °C with 5% CO₂. Medium were changed every day on the apical and basolateral side. On day 4, the apical medium was removed, and basolateral bathing solution exchanged for ALI complete medium (StemCell), followed by additional exchanges three times per week for at least 21 days until reaching a fully differentiated state.