Polymyxin b sulfate

Minimum inhibitory concentration (MIC) was obtained by broth dilution as described (Cuenca-Estrella et al., 2003 (link)), with modifications. Briefly, bacteria and yeast were previously cultured as described above. Microorganisms were then diluted in PBS (0.5 McFarland) and then diluted again (1000-fold) in LB for bacteria or Bacto Tryptic Soy broth (Sigma Aldrich) for yeasts, before seeding a 96-well plate. In each well, 180 μL of this dilution was placed, along with 10 μL polymyxin B sulfate (AppliChem GmbH), ciprofloxacin (Sigma Aldrich) or (R)-(+)-limonene [(+)-p-mentha-1,8-diene,(+)-carvene,(R)-4-isopropenyl-1-methyl-1-cyclohexene] (Sigma Aldrich) (stock prepared in 95% ethanol) to achieve a final known concentration, and 10 μL DBPS. When indicated, the 10 μL DBPS were replaced by 10 μL of purified OMVs to achieve a final known concentration. Alternatively, and when indicated, the 10 μL DBPS were replaced by 10 μL of bacterial supernatant. To obtain the supernatant, bacteria were cultured in LB as stated above (OD₆₀₀ = 1.0–1.3), centrifuged 10 min at 5,400 × g at 4°C, the pellet was discarded, and the supernatant fraction was filtered (0.45 μm). The 96-well plates were incubated overnight at 37°C (bacteria) or 24 h at 28°C (yeasts). The MIC was determined by OD₆₀₀ measurement and corroborated by visual inspection and plating onto agar plates.

Bacterial strains and plasmids used in this work are listed in Table 1. L. monocytogenes Scott A was used as the wild-type (WT) strain and acquired from the International Life Sciences Institute (ILSI) North America [17 (link)]. E. coli DH5α [18 (link)] and S17-1 λpir [19 (link)] were employed as the host for cloning constructs and as donor strain for conjugational plasmid transfer, respectively. L. monocytogenes strains were grown at 30 °C in Brain Heart Infusion (BHI; Oxoid, Hampshire, UK). E. coli strains were grown in Luria-Bertani (LB; 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) at 37 °C. Antibiotics were used when appropriate in the following concentrations: 50 µg/mL erythromycin (Acros Organics, Fair Lawn, NJ, USA) (Ery), 50 µg/mL kanamycin (AppliChem GmbH, Darmstadt, Germany) (Km), 100 μg/mL ampicillin (Thermo Fisher Scientific, Waltham, MA, USA) (Amp), 20 µg/mL polymyxin B sulfate (AppliChem GmbH) and 10 μg/mL chloramphenicol (Acros Organics) (Cm). Other chemicals used in this work include t-CIN (Acros Organics), N-Acetylglucosamine (Sigma-Aldrich, Saint Louis, MO, USA) and isopropyl β-D-1-thiogalactopyranoside (Acros Organics) (IPTG, 1 mM).

Salmonella enterica subsp. enterica sv. Typhi strain STH2370 (S. Typhi) was used as the parental strain (Valenzuela et al., 2014 (link)). The strain was routinely grown in liquid culture using Luria Bertani medium (Bacto tryptone [Gibco], 10 g/L; Bacto yeast extract [Gibco], 5 g/L; NaCl [Winkler], 5 g/L; prepared in distilled water) at 37°C with shaking. When required, the medium was supplemented with agar (15 g/L), ampicillin (Amp, AppliChem GmbH), polymyxin B sulfate (Pmb, AppliChem GmbH), or meropenem (Mer, Sigma Aldrich). For S. Typhi, 1× Amp represents a concentration equivalent to the Minimum Inhibitory Concentration (MIC) of Amp (6.25 μg/mL), while 1× Pmb corresponds to the MIC of polymyxin B (0.31 μg/mL). Various concentrations based on these standards were employed, such as 0.25× Amp (1.56 μg/mL), 0.5× Amp (3.13 μg/mL), 2× Pmb (0.63 μg/mL), and 4× Pmb (1.25 μg/mL).