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 forPseudomonas aeruginosa is the modification of type II topoisomerases (DNA gyrase and topoisomerase IV). We examined the mutations in quinolone-resistance-determining regions (QRDR) ofgyrA, 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 orparE 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 ingyrA and parC, which can be explained by the high inhibitory activities of sitafloxacin against both mutated enzymes.

[1]  M. Ferraro,et al.  Activities of Newer Fluoroquinolones against Streptococcus pneumoniae Clinical Isolates Including Those with Mutations in the gyrA, parC, and parELoci , 1999, Antimicrobial Agents and Chemotherapy.

[2]  E. Bruck,et al.  National Committee for Clinical Laboratory Standards. , 1980, Pediatrics.

[3]  H. Niki,et al.  New topoisomerase essential for chromosome segregation in E. coli , 1990, Cell.

[4]  K. Drlica,et al.  DNA gyrase, topoisomerase IV, and the 4-quinolones , 1997, Microbiology and molecular biology reviews : MMBR.

[5]  T. Nishino,et al.  nfxC-type quinolone resistance in a clinical isolate of Pseudomonas aeruginosa , 1995, Antimicrobial agents and chemotherapy.

[6]  D. Hooper.  Clinical applications of quinolones. , 1998, Biochimica et biophysica acta.

[7]  T. Funato,et al.  [Activities of antimicrobial agents against 5,180 clinical isolates obtained from 26 medical institutions during 1998 in Japan. Levofloxacin--Surveillance Group]. , 2000, The Japanese journal of antibiotics.

[8]  D. Hooper,et al.  Quinolone resistance locus nfxD of Escherichia coli is a mutant allele of the parE gene encoding a subunit of topoisomerase IV , 1997, Antimicrobial agents and chemotherapy.

[9]  S. Nakamura,et al.  Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli , 1990, Antimicrobial Agents and Chemotherapy.

[10]  P. Courvalin,et al.  Characterization of a mutation in the parE gene that confers fluoroquinolone resistance in Streptococcus pneumoniae , 1997, Antimicrobial agents and chemotherapy.

[11]  T. Takenouchi,et al.  Detection of gyrA Mutations among 335Pseudomonas aeruginosa Strains Isolated in Japan and Their Susceptibilities to Fluoroquinolones , 1999, Antimicrobial Agents and Chemotherapy.

[12]  D. Hooper.  Mechanisms of fluoroquinolone resistance. , 1999, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[13]  Y. Onodera,et al.  Quinolone Resistance Mutations in the GrlB Protein of Staphylococcus aureus , 1998, Antimicrobial Agents and Chemotherapy.

[14]  K. Poole,et al.  Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon , 1993, Journal of bacteriology.

[15]  B. Wretlind,et al.  Mechanisms of quinolone resistance in clinical strains of Pseudomonas aeruginosa. , 1998, Microbial drug resistance.

[16]  K. Hoshino,et al.  Contribution of the C-8 substituent of DU-6859a, a new potent fluoroquinolone, to its activity against DNA gyrase mutants of Pseudomonas aeruginosa , 1995, Antimicrobial agents and chemotherapy.

[17]  H. Narita,et al.  DNA gyrase gyrA mutations in quinolone-resistant clinical isolates of Pseudomonas aeruginosa , 1995, Antimicrobial agents and chemotherapy.

[18]  L. Piddock,et al.  A novel gyrB mutation in a fluoroquinolone-resistant clinical isolate of Salmonella typhimurium. , 1995, FEMS microbiology letters.

[19]  L. Bryan,et al.  Cloning and nucleotide sequence of Pseudomonas aeruginosa DNA gyrase gyrA gene from strain PAO1 and quinolone-resistant clinical isolates , 1994, Antimicrobial Agents and Chemotherapy.

[20]  S. Nakamura,et al.  Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli , 1990, Antimicrobial Agents and Chemotherapy.

[21]  R. Hancock.  Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. , 1998, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[22]  T. Tsuchiya,et al.  Expression in Escherichia coli of a New Multidrug Efflux Pump, MexXY, from Pseudomonas aeruginosa , 1999, Antimicrobial Agents and Chemotherapy.

[23]  T. Köhler,et al.  Differential selection of multidrug efflux systems by quinolones in Pseudomonas aeruginosa , 1997, Antimicrobial agents and chemotherapy.

[24]  V. Jarlier,et al.  Role of mutations in DNA gyrase genes in ciprofloxacin resistance of Pseudomonas aeruginosa susceptible or resistant to imipenem , 1995, Antimicrobial agents and chemotherapy.

[25]  K. Poole,et al.  Overexpression of the mexC–mexD–oprJ efflux operon in nfxB‐type multidrug‐resistant strains of Pseudomonas aeruginosa , 1996, Molecular microbiology.

[26]  M. Yasuda,et al.  Mutations in the gyrA and parC genes in fluoroquinolone-resistant clinical isolates of Pseudomonas aeruginosa , 1997, Antimicrobial agents and chemotherapy.

[27]  N. Høiby,et al.  Molecular Mechanisms of Fluoroquinolone Resistance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients , 2000, Antimicrobial Agents and Chemotherapy.

[28]  H. Hiasa,et al.  DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities. , 1998, Biochimica et biophysica acta.

[29]  J. Waitz.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically , 1990 .

[30]  Mary Jane Ferraro,et al.  Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically : approved standard , 2000 .

[31]  N. Gotoh,et al.  Characterization of MexE–MexF–OprN, a positively regulated multidrug efflux system of Pseudomonas aeruginosa , 1997, Molecular microbiology.

[32]  Y. Onodera,et al.  Cloning, Expression, and Enzymatic Characterization of Pseudomonas aeruginosaTopoisomerase IV , 1999, Antimicrobial Agents and Chemotherapy.

[33]  V. Jarlier,et al.  Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of Pseudomonas aeruginosa , 1999, Antimicrobial Agents and Chemotherapy.

[34]  M. Gründling,et al.  A large outbreak of multiresistant Pseudomonas aeruginosa strains in north-eastern Germany. , 1999, The Journal of antimicrobial chemotherapy.

[35]  K. West,et al.  Mutagenesis of E477 or K505 in the B' domain of human topoisomerase II beta increases the requirement for magnesium ions during strand passage. , 2000, Biochemistry.