The major mechanism of resistance to fluoroquinolones for Pseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinoloneresistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolates. There were 150 isolates with reduced susceptibilities to levofloxacin and 127 with reduced susceptibilities to ciprofloxacin among 513 isolates collected during 1998 and 1999 in Japan. Sequencing results predicted replacement of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of the 150 strains (82.7%); among these, 89 isolates possessed mutations in parC or parE which lead to amino acid changes. Substitutions of both Ile for Thr-83 in GyrA and Leu for Ser-87 in ParC were the principal changes, being detected in 48 strains. These replacements were obviously associated with reduced susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; however, sitafloxacin showed high activity against isolates with these replacements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site-directed mutagenesis and compared the inhibitory activities of the fluoroquinolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These results indicated that, compared with other available quinolones, sitafloxacin maintained higher activity against recent clinical isolates with multiple mutations in gyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.

Escherichia coli quinolone-resistant strains with mutations of the parC gene, which codes for a subunit of topoisomerase IV, were isolated from a quinolone-resistant gyrA mutant of DNA gyrase. Quinolone-resistant parC mutants were also identified among the quinolone-resistant clinical strains. The parC mutants became susceptible to quinolones by introduction of a parC+ plasmid. Introduction of the multicopy plasmids carrying the quinolone-resistant parC mutant gene resulted in an increase in MICs of quinolones for the parC+ and quinolone-resistant gyrA strain. Nucleotide sequences of the quinolone-resistant parC mutant genes were determined, and missense mutations at position Gly-78, Ser-80, or Glu-84, corresponding to those in the quinolone-resistance-determining region of DNA gyrase, were identified. These results indicate that topoisomerase IV is a target of quinolones in E. coli and suggest that the susceptibility of E. coli cells to quinolones is determined by sensitivity of the targets, DNA gyrase and topoisomerase IV.

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Two new ditopic ligands, 5,5"-azobis(2,2'-bipyridine) (5,5"-azo) and 5,5"-azoxybis(2,2'-bipyridine) (5,5"-azoxy), were prepared by the reduction of nitro precursors. Mononuclear and dinuclear Ru(II) complexes having one of these bridging ligands and 2,2'-bipyridine terminal ligands were also prepared, and their properties were compared with previously reported Ru(II) complexes having 4,4"-azobis(2,2'-bipyridine) (4,4"-azo). The X-ray crystal structure showed that 5,5"-azo adopts the trans conformation and a planar rodlike shape. The X-ray crystal structure of [(bpy)(2)Ru(5,5"-azo)Ru(bpy)(2)](PF(6))(4) (Ru(5,5"-azo)Ru) showed that the bridging ligand is in the trans conformation and nearly planar also in the complex and the metal-to-metal distance is 10.0 A. The azo or azoxy ligand in these complexes exhibits reduction processes at less negative potentials than the terminal bpy's due to the low-lying pi level. The electronic absorption spectra for the complexes having 5,5"-azo or 5,5"-azoxy exhibit an extended low-energy metal-to-ligand charge-transfer absorption. The ligands, 5,5"-azo and 5,5"-azoxy, and the mononuclear complex, [(bpy)(2)Ru(5,5"-azo)](2+), isomerize reversibly upon light irradiation. The low-energy MLCT state sensitizes the isomerization of the azo moiety in this complex. While [(bpy)(2)Ru(4,4"-azo)Ru(bpy)(2)](PF(6))(4) exhibits light switch properties, namely, significant electrochromism and a large luminescence enhancement, upon reduction, Ru(5,5"-azo)Ru does not show these properties. The radical anion formation upon reduction of these complexes has been confirmed by ESR spectroscopy.