All chemicals were purchased from Fisher Scientific (Atlanta, GA), VWR International (Pittsburg, PA) or Sigma Aldrich (St. Louis, MO). Luria-Bertani (LB) liquid media, prepared from its components (5 g yeast extract, 10 g tryptone and 10 g sodium chloride in 1 L ultra-pure DI water), and Mueller-Hinton (MH) liquid media (21 g premixed MH in 1 L ultra-pure DI water) were used to grow E. coli and P. aeruginosa, respectively. LB agar media (40 g premixed LB agar in 1 L ultra-pure DI water) and MH agar media (38 g premixed MH agar in 1 L ultra-pure DI water) were used to enumerate the colony forming units (CFUs) of E. coli and P. aeruginosa strains, respectively (Keren et al., 2004a (link); Amato et al., 2013 (link); Orman and Brynildsen, 2015 (link)). Phosphate Buffered Saline (PBS) solution was used to wash the cells to remove the chemicals and antibiotics before plating them on agar media. For persister assays, 5 μg/ml ofloxacin and 200 μg/ml ampicillin were used (Keren et al., 2004a (link), b (link); De Groote et al., 2009 (link); Allison et al., 2011 (link)). For selection and retention of plasmids in bacteria, 50 μg/ml kanamycin was added in culture media (Orman and Brynildsen, 2015 (link)). To induce fluorescent protein expression, 1 mM IPTG was used (Orman and Brynildsen, 2015 (link)).
Primary drug screening was performed using Phenotype MicroArrays (PM11-20) in 96-well plate formats, containing various chemicals including FDA approved compounds (Biolog Inc., Hayward, CA). Eleven chemicals, identified as initial hits, were purchased separately for further investigation: amitriptyline hydrochloride (Fisher catalog# 50-144-4347), trifluoperazine hydrochloride (Fisher catalog# T28495G), thioridazine hydrochloride (Fisher catalog# 30-705-0), CPZ (Fisher catalog# C24815G), CCCP (Fisher catalog# 04-525-00), protamine sulfate (Fisher catalog# AAJ6292609), promethazine hydrochloride (Fisher catalog# P2029100G), dodecyltrimethyl ammonium bromide (Fisher catalog# D146825G), triclosan (Fisher catalog# 64-795-01GM), polymyxin B Sulfate (Fisher catalog# 52-915-GM) and poly-L-lysine hydrochloride (VWR catalog# IC15269080). All chemicals were dissolved in ultra-pure DI water followed by filter-sterilization, except for CCCP and triclosan which were dissolved in DMSO. All LB and MH media were sterilized by autoclaving. Overnight pre-cultures were prepared in 14-ml falcon tubes containing 2 ml LB broth inoculated from a 25% glycerol (−80°C) cells stock and grown for 24 h at 37°C with shaking (250 rpm). Overnight pre-cultures were diluted in fresh 2 ml media in 14-ml test tubes or 25 ml media in 250-ml baffled flasks for the subsequent assays as described below. Cells cultured in the presence of the solvent (DI water or DMSO) served as controls when the cultures were treated with chemical inhibitors.
Primary drug screening was performed using Phenotype MicroArrays (PM11-20) in 96-well plate formats, containing various chemicals including FDA approved compounds (Biolog Inc., Hayward, CA). Eleven chemicals, identified as initial hits, were purchased separately for further investigation: amitriptyline hydrochloride (Fisher catalog# 50-144-4347), trifluoperazine hydrochloride (Fisher catalog# T28495G), thioridazine hydrochloride (Fisher catalog# 30-705-0), CPZ (Fisher catalog# C24815G), CCCP (Fisher catalog# 04-525-00), protamine sulfate (Fisher catalog# AAJ6292609), promethazine hydrochloride (Fisher catalog# P2029100G), dodecyltrimethyl ammonium bromide (Fisher catalog# D146825G), triclosan (Fisher catalog# 64-795-01GM), polymyxin B Sulfate (Fisher catalog# 52-915-GM) and poly-L-lysine hydrochloride (VWR catalog# IC15269080). All chemicals were dissolved in ultra-pure DI water followed by filter-sterilization, except for CCCP and triclosan which were dissolved in DMSO. All LB and MH media were sterilized by autoclaving. Overnight pre-cultures were prepared in 14-ml falcon tubes containing 2 ml LB broth inoculated from a 25% glycerol (−80°C) cells stock and grown for 24 h at 37°C with shaking (250 rpm). Overnight pre-cultures were diluted in fresh 2 ml media in 14-ml test tubes or 25 ml media in 250-ml baffled flasks for the subsequent assays as described below. Cells cultured in the presence of the solvent (DI water or DMSO) served as controls when the cultures were treated with chemical inhibitors.
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