Antimicrobial Effects on E. coli

For all experiments performed with cells in exponential phase, E. coli overnight cultures were diluted 1:250 in 25mL of Luria-Bertani (LB) media and grown to an OD_600nm of 0.3 in 250 mL flasks at 37 °C, 300 rpm, and 80% humidity. All antimicrobial treatments were performed in 500 μL samples in 24-well plates incubated at 37 °C, 900 rpms, and 80% humidity. For experiments with bacterial persister cells, E. coli were grown to stationary phase for 16 h at 37 °C, 300 rpm, and 80% humidity in 25 mL of LB. Cells were then treated with 5 μg/mL ofloxacin for 4 h to kill non-persister cells. The samples were then washed with PBS and suspended in M9 minimal media and treated with the different antibiotics to determine killing of persisters. For experiments with biofilms, an E. coli culture grown overnight was diluted 1:200 into MBEC Physiology and Genetic Assay wells (MBEC BioProducts, Edmonton, Canada) and grown for 24 h at 30 °C, 0 rpm and 80% humidity. All wells containing biofilms were then treated with the different antibiotics. After treatment, the wells were washed with PBS 3x and then sonicated for 45 min in order to disrupt the biofilm and plate cells to count colony-forming units (cfu). Unless otherwise specified, the following concentrations were used in the E. coli antimicrobial treatments: 10, 20, 30, 60 and 120 μM silver nitrate, 0.25 μg/mL and 5 μg/mL gentamicin, 1 μg/mL and 10μg/mL ampicillin, 0.03 μg/mL and 3 μg/mL ofloxacin, and 30 μg/mL vancomycin. Kill curves for the antimicrobial treatments were obtained by spot-plating serially diluted samples and counting cfu. Gene knockout strains were constructed by P1-phage transduction from the Keio knockout mutant collection. Raw data (cfu/mL) for killing assays for all strains are in table S2. Construction of the genetic reporter strains for iron misregulation, superoxide production and disulfide bond formation, as well as the sodA overexpression strain, was performed using conventional molecular cloning techniques. The fluorescent reporter dye 3'-(p-hydroxyphenyl fluorescein (HPF) was used as previously described (18 (link)) at 5 mM to detect hydroxyl radical (OH•) formation. The fluorescent dye, propidium iodide (PI), was used at concentrations of 1 mM to monitor membrane permeability. Fluorescence data were collected using a Becton Dickinson FACSCalibur flow cytometer. For the permeability and OH• production assays, fluorescence of the respective dyes was determined as a percent change using the following formula: ((Fluorescence_dye – Fluorescence_{no dye})/(Fluorescence_{no dye}))*(100). For the OH• quenching experiments, cells were treated with 150mM thiourea and AgNO₃ simultaneously. Release of protein-bound iron in an E. coli cell lysate was detected by incubating samples for 1 h in a 10 mM Ferene-S assay and measuring absorbance at 593 nm. The lysates were prepared by sonication in 20 mM Tris/HCl pH 7.2 buffer. The lysates were treated either with heat (90 °C for 20 min) or AgNO₃ (30 μM for 1 h). All samples analyzed with the Jeol 1200EX – 80kV transmission electron microscope were fixed utilizing glutaraldehyde, dehydrated using ethanol, embedded using spur resin, and microtomed in ~60 nm thickness samples. Mouse experiments were performed with male Charles River mice as described in the main text and the in vivo studies section below.