Tetracycline

OrthoList RNAi plates were replicated in LB agar, and bacteria were grown overnight at 37 °C. The advantage of growing the bacteria in solid media is the ease of visualising the clones where the bacteria did not grow. If needed, the samples can be kept at 4 °C for 2 days maximum. Next day, we inoculated the RNAi library in 2.2-ml 96-well plates (VWR). Using the pin replicator, we inoculated the bacteria from the LB agar into 1.2 ml of LB supplemented with ampicillin (100 μg/ml, Sigma-Aldrich) and tetracycline (15 μg/ml, Sigma-Aldrich). Positive and negative controls were added in the last column of the plate. We cultured the bacteria overnight at 37 °C with shaking (180 rpm, New Brunswick™ Innova® 44/44R). In order to have fresh cultures, on the day of sorting, we inoculated 100 μl of the O/N cultures in 900 μl of LB supplemented with ampicillin and tetracycline in deep well plates (VWR) and incubated for 3 h at 37 °C with shaking. We added isopropylthio-β-galactoside (IPTG, 1 mM, Sigma-Aldrich) to the wells to induce the expression of the plasmid for 2 h at 37 °C with shaking. Then, we harvested the cultures by centrifugation (10 min, 3200 g, 4 °C, Eppendorf 5810R) and re-suspended the pellets in 250 μl of S-medium supplemented with carbenicillin (25 μg/ml, Sigma-Aldrich), IPTG (1 mM, Sigma-Aldrich) and cholesterol (5 μg/ml, Sigma-Aldrich).

We performed phenotypic antimicrobial susceptibility testing to investigate the occurrence of additional clinically relevant resistance phenotypes. Briefly, a single colony was picked and streaked on chromogenic agar plates containing antibiotics at their clinical breakpoint concentrations according to the published breakpoints of the Clinical and Laboratory Standards Institute [72 ]. We used the antibiotics tetracycline (16 μg/mL; VWR International, Radnor, PA, USA), gentamicin (2 μg/mL; Carl Roth, Karlsruhe, Germany), and ciprofloxacin (1 μg/mL; VWR International, Radnor, PA, USA). The minimum inhibitory concentration of colistin was determined using the MICRONAUT MIC-Strip Colistin Assay (Merlin Diagnostika, Bornheim, Germany) according to the manufacturer’s instructions.

The derivatives of Salmonella enterica serovar Typhimurium strain 14028s and Escherichia coli, as well as the plasmids used in this study, are listed in Tables S2 and S3. Deletion mutants were constructed using the λ-Red homologous recombination system^{45 (link)}. Pfu ultra high-fidelity DNA polymerase (cat. # 600382-51, Agilent, Santa Clara, CA) was used to perform genetic mutagenesis using primers described in Table S4. Bacteria were grown in LB broth at 37 °C in a shaker incubator. Penicillin (cat. # J63032, AlfaAesar), chloramphenicol (cat. # 0230, Amresco), tetracycline (cat. # 0422, Amresco), and kanamycin (cat. # K-120, Goldbio) were added at the final concentrations of 250, 40, 20, or 50 μg/ml, respectively.