Colistin

Antibacterial activity was determined according to Tang et al. (20 (link)). dTHP-1 cells were seeded in 6 well plates and primed with DCATH-2 or medium as described. Log-phase culture of Salmonella enterica subsp. enterica serovar enteritidis 706 (Se706) was added to each well at a MOI of 1. After 2 h incubation at 37°C, cells were washed twice with warm Dulbecco’s Phosphate buffered saline (DPBS; Life technologies) and further incubated for 1 h at 37°C with IMDM/FBS medium containing 300 µg/ml colistin (MP biochemicals, Santa Ana, CA, USA). After incubation, cells were washed 3 times in DPBS and the lysed with 1% triton X-100. Well contents were serially diluted in tryptone soy broth (Oxoid, Basingstoke, UK), plated on tryptone soy agar and counted after 24 h at 37°C.

MICs were determined using the broth microdilution method, following the CSLI guidelines [48 ]. The tests were conducted in ISO-Sensitest Broth (Oxoid, Hamsphire, UK), according to recommendations of the International Standard ISO 20776-1. Standard antibiotics were also included in the assays in order to compare their activity and efficacy. In particular, ciprofloxacin, ampicillin, vancomycin, trimethoprim-sulfamethoxazole (Sigma Aldrich, Madrid, Spain), and colistin (MPBiomedicals, Eschwege, Germany) were investigated.
Briefly, broth microdilution was performed in polystyrene 96-well round bottomed plates in a final volume of 100 μL. Serial two-fold dilutions of the antimicrobial ranging from 128 mg/L to 0.03 mg/L were used. In addition, wells with bacteria alone and with growth media alone were included as positive and negative controls, respectively. As for the inoculum, after an overnight incubation in agar plates, bacterial colonies were resuspended in 0.9% NaCl to reach a 0.5 McFarland (equivalent to 1.5 × 10⁸ CFU/mL) and diluted to obtain a final concentration in the well of 5 × 10⁵ CFU/mL. Finally, the plates were incubated for 18 h at 37 °C. MIC values were expressed in mg/L. We performed three technical and three biological replicates for each agent.