aeruginosa isolates collected from CF patients using a method adopted from Jacob 1954 . Bacterial cells of dense LB cultures (48 h old) of PA01 and PA14 were spun down in a centrifuge. Supernatant was collected and filtered (0.20 μm) and serially Ribociclib diluted in minimal salts medium . For the assay asking if proteinaceous compounds are responsible for killing, the sterile supernatant was heated for 15 s at 100°C. 0.5 ml of a dense culture of a natural isolate was added to 3 ml of molten semi solid LB (bacto-tryptone 10 g, yeast extract 5 g, NaCl 10 g, agar 7 g, 1000 ml dH2O) and poured out over the surface of a Petri dish containing
minimal salts agar (as described above with 10 g of agar added) as an overlay. A dilution series of either non-heat treated or heat treated sterile supernatant was spotted on top of the layer of natural isolate, up to 11 spots of 15 μl were spotted on a single Petri dish with overlay. Cultures were incubated for 48 h at 37°C. The inverse of the highest dilution of sterile supernatant giving rise to inhibition was defined as the inhibition score, which is effectively a measure of the minimal inhibitory concentration (MIC). SAHA HDAC molecular weight The inhibition scores were log transformed
prior to analysis. No inhibition was observed when spotting sterile PA01or PA14 supernatant on top of an overlay with the same strain. To exclude the possibility that bacteriophage are responsible for the observed inhibition, we performed a one-step growth assay as follows. The zones of inhibition formed on overlays of clinical isolates were transferred to an exponential liquid culture of growing cells of the same clinical isolate. After 24 h, cell free extract was prepared and spotted
onto a layer of the clinical isolate. A resulting clear zone of inhibition would be indicative of the presence of bacteriophage because a 24 h liquid culture of clinical isolate should contain even more bacteriophage than the initial culture since bacteriophage are able to reproduce with the appropriate host cells present. We found no evidence for the presence of bacteriophage. Acknowledgements We thank anonymous reviewers and Andy Gardner for comments Tacrolimus (FK506) on this work and Tracy Giesbrecht, Talía Malagón and Danna Gifford for assistance. The Ontario Provincial Government, the Canadian Cystic Fibrosis Foundation and the National Research Council of Canada provided funding. Electronic supplementary material Additional file 1: Table S1. Inhibition of clinical isolates by toxins in cell free extract collected from laboratory strains PA01 and PA14 as a function of metabolic similarity (correlation coefficient) between toxin producer and clinical isolate based on BIOLOG profiles. A unimodal non-linear relationship peaking at intermediate metabolic similarity give best fit to the data for producer PA14 (solid lines), better than a linear fit; for PA01 no such relationship was found.