Bacterial Strain and Media
The bacterial strain used in this study was Bacillus oleronius strain ATCC 700005 (ATCC, Manassas, VA, USA). Bacteria were cultured in Muller Hinton II (MHII) broth at 37°C (Becton Dickson and Company, Sparks, MD, USA).
Broth Microdilution Assay
Minimum inhibitory concentrations (MIC) for bacitracin, CAS 1405-87-4 (Acros Organics, Fair Lawn, NJ, USA), clindamycin, CAS 58207-19-5 (Tokyo Chemical Industry CO., Tokyo, Japan), neomycin, CAS 1405-10-3 (Fisher BioReagents, Fair Lawn, NJ, USA), and prodigiosin, CAS 82-89-3 (AdipoGen, San Diego, CA, USA) were determined using the broth microdilution assay described by the Clinical and Laboratory Standards Institute. Briefly, a 1:2 dilution series of prodigiosin was prepared in a 96-well microtiter plate. Overnight cultures of B. oleronius were diluted in MHII broth and inoculated to a final concentration of 5×104 CFU/well. The total volume in each microtiter well was 100 μL. For the prodigiosin experiment, the concentration of ethanol was maintained at 0.8% (v/v) in all conditions. MICs were determined by visual inspection following 16–18 h of static incubation at 37°C. Using unaided eye observation, we determined the MIC to be the lowest concentration of a compound that completely inhibited bacterial growth. The MIC values reported here represent the average of 4 independent experiments for prodigiosin and clindamycin and the average of 5 independent experiments for bacitracin and neomycin. The MIC values are reported in the following format: average (SD).
Growth Curve with Prodigiosin Addition
An overnight culture of B. oleronius was diluted 1:100 into fresh MHII broth and divided into 7 mL cultures. The freshly diluted cultures were grown at 37°C with shaking at 200 rpm. Bacterial growth was monitored by measuring optical density at 650 nm (OD650) using a Spectronic 20D (Milton Roy, Dallas, TX, USA). For the first 180 min, OD650 was measured at 60 min intervals. After 195 min of growth, either prodigiosin, an equivalent volume of ethanol, or plain MHII broth, was aseptically added to the cultures. The tubes were then returned to the shaking incubator for 30 min prior to the next OD650 measurement. Subsequently, culture density at OD650 was measured at 30 min intervals for an additional 210 min. The concentration of ethanol was maintained at 0.05% (v/v) in prodigiosin and ethanol control conditions. The growth curves reported here represent the average of 3 independent experiments.