Type II topoisomerase mutations in fluoroquinolone-resistant clinical strains of Pseudomonas aeruginosa isolated in 1998 and 1999: Role of target enzyme in mechanism of fluoroquinolone resistance

The major mechanism of resistance to fluoroquinolones for Pseudomonas aerug inosa is the modification of type II topoisomerases (DNA gyrase and topoiso merase IV), We examined the mutations in quinolone-resistance-determining r egions (QRDR) of gyrA, gyrB, parC, and parE genes of recent clinical isolat es. There were 150 isolates with reduced susceptibilities to levofloxacin a nd 127 with reduced susceptibilities to ciprofloxacin among 513 isolates co llected during 1998 and 1999 in Japan, Sequencing results predicted replace ment of an amino acid in the QRDR of DNA gyrase (GyrA or GyrB) for 124 of t he 150 strains (82.7%); among these, 89 isolates possessed mutations in par C or parE which lead to amino acid changes. Substitutions of both Ile for T hr-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 r educed susceptibilities to levofloxacin, ciprofloxacin, and sparfloxacin; h owever, sitafloxacin showed high activity against isolates with these repla cements. We purified GyrA (The-83 to Ile) and ParC (Ser-87 to Leu) by site- directed mutagenesis and compared the inhibitory activities of the fluoroqu inolones. Sitafloxacin showed the most potent inhibitory activities against both altered topoisomerases among the fluoroquinolones tested. These resul ts indicated that, compared,vith other available quinolones, sitafloxacin m aintained higher activity against recent clinical isolates with multiple mu tations in gyrA and parC, which can be explained by the high inhibitory act ivities of sitafloxacin against both mutated enzymes.