6]) and 70

μL of the suspension was mixed with an equal amount of 1.6% Lapatinib low melt agarose (Cambrex, East Rutherford, NJ). This mixture was pipetted into a plug mold (Bio-Rad, Hercules, CA) and allowed to solidify at room temperature. Plugs were added to plug lysis solution (1 M NaCl, 100 mM EDTA [pH 7.5], 0.5% Brij-58, 0.5% Sarcosyl, 0.2% Deoxycholate, 6 mM Tris-HCl [pH 7.6], 1 mg/mL Lysozyme powder, 20 μg/ml RNase) and incubated for 4 h at 37°C with shaking. Plugs were then placed in Proteinase K solution (0.5 M EDTA [pH 9-9.5], 1% Sarcosyl, 50 μg/ml Proteinase K) and incubated overnight at 50°C with shaking. Plugs were washed 3-4 times with TE buffer (10 mM Tris-HCl [pH 7.5], 0.1 mM EDTA [pH 7.5]) at 37°C and then stored at 4°C. DNA in a 2-3 mm piece of the gel plug was restricted https://www.selleckchem.com/products/Temsirolimus.html using 20 U SpeI (New England Biolabs, Ipswich, MA) in a reaction volume of 0.2 mL at 37°C. The digestion products were melted and electrophoresis

was performed on a 1.0% agarose gel, in 0.5X TBE (VWR International Ltd, Mississauga, ON), using a CHEF DR III apparatus (Bio-Rad, Hercules, CA). Electrophoresis conditions were as follows: 20 h at 6 V/cm with switch times of 5 s to 45 s with a linear ramping factor. Using the ladder, all banding patterns were inspected for the presence/absence of a visible band at 51 locations. These presence/absence data were used to calculate the genetic distance by calculating the Jaccard similarity (Jaccard distance equals 1- Jaccard similarity) of natural isolates to both laboratory strains PA01 and PA14: where Mij represents the total number of positions where bands are present Afatinib supplier (i = j = 1), or when one strain or the other possesses a band (i ≠ j). Other measures of similarity such as the Hamming distance, Dice coefficient and correlation coefficient gave similar qualitative results. We used R software (version 2.6.1) to calculate distance measures and for all statistical analyses. Estimation of metabolic similarity Resource use was measured using BIOLOG GN2 plates that consist of different wells with a total of 95 different carbon sources. All 55 clinical isolates and strains P. aeruginosa PA01 and PA14 were grown

up in liquid LB medium. From a dense stationary phase culture, 20 μl was added to 20 ml of a minimal salts medium (Na2HPO4 6.7 g, KH2PO4 3 g, NaCl 0.5 g, NH4Cl 1.0 g, 1000 ml dH2O) which was used to inoculate the Biolog plates after a 2 h starvation period. For clinical isolates, 1 Biolog plate was used, for P. aeruginosa PA01 and PA14 three replicate plates were used. Right after inoculation and after 48 h of incubation at 37°C, the OD (590 nm) was measured of all wells. The difference in OD at the two time points is a measure of how well a given strain is able to use a given resource. To quantify the metabolic similarity, we calculated the correlation coefficient between the OD values of the different strains. Inhibition assays The strains P.