The results indicate it is essential to evaluate antimicrobial strategies over a range of perturbations relevant to the targeted application so that accurate predictions regarding efficacy can be made. Methods Bacterial strains and growth conditions E. coli K-12 MG1655 gene deletion mutants were constructed using the KEIO mutant library and P1 transduction techniques

[50, 51]. E. coli cultures were grown in low salt Luria-Bertani (LB) broth with or without different substrate TSA HDAC mouse supplements. When added, the supplements were autoclaved separately from the LB medium. The average starting pH of the medium was 6.8. All antibiotics were utilized at a final concentration of 100 ug/ml. The tested antibiotics had different molecular weights so this mass concentration represents a different molar concentration for each agent. Culturing temperatures ranged from 21 to 42°C depending on experiment. Colony biofilm culture antibiotic tolerance testing The colony biofilm culturing Bcl-2 inhibitor method has been described previously [3, 4, 7, 52, 53]. Briefly, colony biofilm systems consist of agar plates, sterile 0.22 μm pore- 25 mm diameter polycarbonate membranes (GE Water and Process Technologies,

K02BP02500), and the desired bacterial strains. The membrane is placed aseptically on agar plates and inoculated with 100 uL of an exponentially growing culture (diluted to OD600 = 0.1). The culture is grown for 6 hours on untreated plates of the desired medium composition. After the initial growth phase, the biofilm is aseptically transferred Phospholipase D1 to either a treated or a control plate where it is incubated for an additional 24 hours. The nutrients and antibiotics enter the biofilm

from below the membrane. Antibiotic penetration of colony biofilms has been studied expensively suggesting the agent readily moves throughout the biofilm [3]. The delivery of antibiotic is Forskolin datasheet diffusion based analogous to the many antibiotic impregnated coating systems. After treatment, the colony biofilms are aseptically transferred to 10 ml glass test tubes pre-filled with 5 mL of sterile phosphate buffered saline. The colony biofilm is vortexed vigorously for 1 minute to separate the cells from the membrane. The membrane is removed and discarded. The dislodged biofilm is homogenized using a tissue homogenizer for 40 seconds to ensure complete physical disaggregation. The homogenized culture is serially diluted and colony forming units (cfu’s) per membrane are enumerated using the drop-plate method [54]. Planktonic culture antibiotic tolerance testing For planktonic antibiotic tolerance experiments, 50 ml cultures were grown exponentially for six hours with shaking (250 ml flask, 150 rpm) at 37°C in untreated medium (with or without 10 g/L glucose). The cells were collected using centrifugation (800 rcf, 20 minutes).