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

Abstract

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.

Phenotypic and Molecular Characterization of Acinetobacter Clinical Isolates Obtained from Inmates of California Correctional Facilities

“…Resistance to FQs in Gram-negative bacteria primarily results from point mutations in gyrA and/or parC gene sequences that render significantly lower drug affinity for DNA gyrase (containing mutated GyrA) and DNA topoisomerase (composed of mutated ParC) (1,57,65). In A. baumannii, the most common amino acid substitutions occur at position 83 of GyrA and position 80 of ParC (31,72,73,76).…”

Antibacterial-ResistantPseudomonas aeruginosa: Clinical Impact and Complex Regulation of Chromosomally Encoded Resistance Mechanisms

“…Similar to the case for other gram-negative bacteria, DNA gyrase is the primary target for the fluoroquinolones in P. aeruginosa (84). Therefore, the first target-specific mutations are typically observed within the quinolone resistance determining region (QRDR) of gyrA (6,116,165,249,283). The highest levels of resistance are observed in strains that have mutations in the QRDR of both gyrA and the topoisomerase IV gene parC (6,90,91,116,165).…”

“…Therefore, the first target-specific mutations are typically observed within the quinolone resistance determining region (QRDR) of gyrA (6,116,165,249,283). The highest levels of resistance are observed in strains that have mutations in the QRDR of both gyrA and the topoisomerase IV gene parC (6,90,91,116,165). Although mutational changes within the other two genes, gyrB and parE, have been described, the prevalence of these mutations appears to be much lower (6).…”

Phenotypic and Molecular Characterization ofAcinetobacter baumanniiClinical Isolates from Nosocomial Outbreaks in Los Angeles County, California

“…A major mechanism of fluoroquinolone resistance in gramnegative bacteria involves changes in the structure (and hence in the affinity to the drugs) of the drug targets DNA gyrase (encoded by the gyrA and gyrB genes) and DNA topoisomerase IV (encoded by the parC and parE genes) (1,35,45). Specifically, amino acid substitutions at certain positions in subunits A (GyrA and ParC) of both DNA gyrase and DNA topoisomerase IV, due to point mutations in the QRDRs of the genes encoding these two polypeptides, have been found to contribute to fluoroquinolone resistance.…”