Routes of quinolone permeation in Pseudomonas aeruginosa were investigated by using sparfloxacin as a prototype compound. [14C]sparfloxacin cell labeling was 13 to 28% lower in three protein D2-deficient mutants resistant to imipenem than in their imipenem-susceptible counterparts. In four impermeability-type quinolone-resistant strains isolated from pefloxacin-treated animals, we observed two- to fourfold-greater resistance to imipenem, reduced protein D2 expression in the outer membrane according to Western blotting (immunoblotting), and 25 to 29% decreased cell labeling with imipenem. In a protein D2-producing strain but not in its protein D2-deficient isogenic mutant, uptake of [14C]sparfloxacin was strongly inhibited by L-lysine and imipenem, which act as substrates for protein D2. Conversely, binding of [14C]imipenem in a porin D2-positive strain was reduced by sparfloxacin but not by the nonamphoteric quinolone nalidixic acid. Sparfloxacin, imipenem, and lysine possess a carboxyl group and a potentially protonated nitrogen separated from each other by 0.64 to 1.07 nm as calculated by computer. Hence, protein D2 may catalyze facilitated diffusion for sparfloxacin, as it does for imipenem. In addition, pefloxacin-selected isolates contained 41 to 113% more 3-deoxy-D-mannooctulosonic acid than their quinolone-susceptible counterparts, with MIC increases of 2- to 4-fold for WIN-57273 (n-octanol-phosphate buffer partition coefficient, 13.139), 4- to 8-fold for difloxacin (partition coefficient, 3.093) and sparfloxacin (partition coefficient, 0.431), and 8- to 16-fold for norfloxacin (partition coefficient, 0.059) and ciprofloxacin (partition coefficient, 0.056). Thus, we hypothetize that in quinolone-selected strains, increased amounts of lipopolysaccharide form a permeability barrier that acts preferentially against hydrophilic quinolones
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