After 16 h, the samples were then centrifuged at 12000 × g for 5 min at room temperature and the fluorescence of the supernatant Cell Cycle inhibitor was measured using the excitation and emission wavelengths
of 295 and 490 nm, respectively. Levofloxacin concentrations were calculated using a standard curve of the antibiotic (concentration ranging from 0.42 μg/ml to 6.38 μg/ml) in 0.1 M glycine-HCl buffer, pH 3.0. To correct for any endogenous signal the fluorescence of a control cell lysate, measured on samples not exposed to the drug, was subtracted from the experimental values. The intracellular levels of levofloxacin were expressed as drug accumulation in 109 cells, after counting of viable cells for each time point. The accumulation of levofloxacin was determined at the following time intervals: 0 min, 0 min+ drug, 2.5 min, 5 min, 10 min, 15 min, and 20 min. To determine
whether levofloxacin was actively effluxed from B. cenocepacia J2315 and the mutant strains, reserpine (8 μg/ml) was added 2.5 GSK126 supplier min after the addition of levofloxacin and the samples were treated as described above. Purification, detection and quantification of N-acyl homoserine lactone (AHLs) The purification, detection and visualization of AHL signal molecules from culture supernatants were performed as described previously . Bacterial strains were inoculated in 50 ml of half diluted LB and grown at 37°C with constant agitation until OD600 reached 2.5. Organic extractions with ethyl acetate (0.1% acetic acid) were performed twice on each supernatant and extracts were dried and resuspended in CH5424802 purchase acidified ethyl acetate in 1/1000 of the original volume. Quantification of AHLs was determined using the reporter plasmid pSCR1. This plasmid
contains the cepR gene and the cepI gene promoter controlling the expression of a promoterless β-galactosidase (lacZ) gene and functions as a sensor of AHL molecules . Overnight cultures of E. coli DH5α Fluorometholone Acetate carrying pSCR1 were normalized to an OD600 of 0.1 in a volume of 20 ml LB containing 10 μL of the AHL purified extract (prepared as described above). 10 μL of ethyl acetate were used as negative control, while 100 nM of synthetic C8-HSL (Sigma-Fluka) was used as positive control. Cultures were then grown with agitation at 37°C for 6 h and β-galactosidase activities were determined . Acknowledgements The authors are grateful to Dr. Claudio Seppi (Dipartimento di Biochimica A. Castellani, University of Pavia, Italy) for fluorometer availability to perform efflux experiments. R.S.F. was supported by a studentship from the Canadian Cystic Fibrosis Foundation. M.A.V. holds a Canada Research Chair in Infectious Diseases and Microbial Pathogenesis. This research was supported by a grant from Italian Cystic Fibrosis Research Foundation (FFC). The project was adopted by FFC Delegation of Lago di Garda e Bergamo. References 1.