Objective To evaluate the role of outer membrane protein (Omp) F-deficiency and active efflux in the accumulation of hydrophilic fluoroquinolones ciprofloxacin (CPLX) and lomefloxacin (LMLX) in resistant E. coli strains. Methods Fluoroquinolone accumulation in bacteria and the effect of active efflux were measured by a fluorescence method. The outer membrane proteins of the bacteria were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). E. coli strains in this study included control strains JF701 and JF703 that are OmpC- or OmpF-deficient mutants of E. coli K-12, respectively, and the fluoroquinolone susceptible strain the fluoroquinolone susceptible strain of Escherichia coli (Ecs) and its in vitroselected resistant strains R2 and R256, and the clinical resistant isolates R5 and R6. Results The steady-state accumulation concentration of each drug in Ecs appeared to be the same as in JF701, while in the OmpF- deficient strain JF703, it was 1/5 CPLX or 1/2 LMLX lower than that in JF701, but JF703 was still susceptible to fluoroquinolones. On the other hand, compared with susceptible strains, a 2- to 10-fold decrease in the accumulation of each drug was found in the resistant strains except R2, in which the accumulation was slightly higher than in JF703. After the addition of 2,4-dinitrophenol (DNP), accumulation of each drug increased, especially in resistant strains, indicating that the function of the active efflux (pump) system in these bacteria had been enhanced dramatically. Furthermore, both OmpF and OmpC in Ecs, OmpF-deficiency in R2 and R256 and OmpC-deficiency in R5 and R6 were observed.Conclusion The decreased accumulation of hydrophilic fluoroquinolones in E. coli involved OmpF-deficiency and active efflux (pump), and the latter may be an important factor.

Objective: To describe the expression and immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections. Methods: The hybrid protein OprF (aa 190-342)-Opr I (aa 21-83) was modified N-terminally, either with a minimal histidine tag or with a homologous sequence of OprF. Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions. Results :This produced three suitable candidates for a vaccination trial: protein His-F- I , which was purified in its native as well as in its refolded form,and the native purified N-terminally extended protein ex-F- I . In mice, significantly higher antibody titers and survival rates after challenge with P. aeruginosa were observed, following immunization with protein His-F- I purified under native conditions. Conclusion: A hybrid OprF-Opr I molecule was cloned and a purification method which yields a protective vaccine against P. aeruginosa infections was established.