A new type of norfloxacin-resistant mutant of Pseudomonas aeruginosa PAO was isolated. This mutant showed cross resistance to imipenem and chloramphenicol and hypersusceptibility to ,-lactam and aminoglycoside antibiotics. The new norfloxacin resistance gene nfxC was mapped near catA (46 min) on the PAO chromosome. Norfloxacin accumulation was decreased in the nfxC mutant; furthermore, the rate of imipenem diffusion through the outer membrane of the nfxC mutant was lower than that of the parent strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins showed a decrease of a 46-kilodalton protein and an increase of a 50-kilodalton protein in the nfxC mutant. We conclude the nfxC is a new norfloxacin resistance gene that affects outer membrane permeability to quinolones and other antimicrobial agents.Quinolone resistance in gram-negative bacteria involves alterations in DNA gyrase and in the likely permeability of the outer cell membrane. Altered DNA gyrases have been reported in quinolone-resistant strains such as Escherichia coli, Citrobacter freundii, Pseudomonas aeruginosa, and Serratia marcescens (1,2,5,6,14,15,28). In E. coli, alteration in cell membrane permeability to quinolones was found to be associated with a decrease in the OmpF porin protein. Such a quinolone-resistant E. coli strain showed cross resistance to unrelated antimicrobial agents such as chloramphenicol and cefoxitin (11,14). Similar resistance mechanisms were found in other members of the family Enterobacteriaceae, such as C. freundii, Klebsiella pneumoniae, and S. marcescens (1,7,10,27).In studying the mechanisms of resistance to norfloxacin in P. aeruginosa PAO strain, we isolated two norfloxacinresistant mutations (nfxA and nfxB) (12). The nfxA gene was shown to be an allele of nalA that encoded the A subunit of DNA gyrase, which is a target enzyme of quinolones. nfxB is a novel mutation that has an alteration in its outer membrane permeability to quinolones, and its resistance is associated with the appearance of a 54-kilodalton (kDa) outer membrane protein. The nfxB mutant is characteristic in that it shows hypersusceptibility to B-lactam and aminoglycoside antibiotics. It has also been reported that other quinoloneresistant mutants (nalB and cfxB) whose cell permeabilities are affected show cross resistance to carbenicillin, chloramphenicol, and tetracycline (25, 26).More recently, we isolated a new type of spontaneous norfloxacin-resistant mutant of a P. aeruginosa PAO strain. This mutant shows cross resistance to imipenem and chloramphenicol, but not to other ,-lactams, and this mutation is genetically distinct from other quinolone-resistant mutations, nalA (cfxA and nfxA), nalB (cfxB), and nfxB.Here we report the biochemical and genetic properties of this novel norfloxacin-resistant mutant of P. aeruginosa PAO.* Corresponding author. MATERIALS AND METHODSBacterial strains and phage. The P. aeruginosa strains used in this study are listed in Table 1. KH 4014a is a spontaneous norfloxacin-resistant...
The antibacterial activities and target inhibition of 15 quinolones against grlA and gyrA mutant strains were studied. The strains were obtained from wild-type Staphylococcus aureus MS5935 by selection with norfloxacin and nadifloxacin, respectively. The antibacterial activities of most quinolones against both mutant strains were lower than those against the wild-type strain. The ratios of MICs for the gyrA mutant strain to those for the grlA mutant strain (MIC ratio) varied from 0.125 to 4. The ratios of 50% inhibitory concentrations (IC 50 s) of quinolones against topoisomerase IV to those against DNA gyrase (IC 50 ratios) also varied, from 0.177 to 5.52. A significant correlation between the MIC ratios and the IC 50 ratios was observed (r ؍ 0.919; P < 0.001). These results suggest that the antibacterial activities of quinolones against the wild-type strain are involved not only in topoisomerase IV inhibition but also in DNA gyrase inhibition and that the target preference in the wild-type strain can be anticipated by the MIC ratios. Based on the MIC ratios, the quinolones were classified into three categories. Type I quinolones (norfloxacin, enoxacin, fleroxacin, ciprofloxacin, lomefloxacin, trovafloxacin, grepafloxacin, ofloxacin, and levofloxacin) had MIC ratios of <1, type II quinolones (sparfloxacin and nadifloxacin) had MIC ratios of >1, and type III quinolones (gatifloxacin, pazufloxacin, moxifloxacin, and clinafloxacin) had MIC ratios of 1. Type I and type II quinolones seem to prefer topoisomerase IV and DNA gyrase, respectively. Type III quinolones seem to target both enzymes at nearly the same level in bacterial cells (a phenomenon known as the dual-targeting property), and their IC 50 ratios were approximately 2.Quinolone antibacterial agents have potent activities against gram-positive and -negative bacteria, and they are currently used for the therapeutic treatment of various bacterial infections. Antibacterial activities of quinolones are involved in their inhibitory activities for DNA gyrase and topoisomerase IV (1,6,14). Both enzymes are members of the type II topoisomerase family that controls bacterial DNA topology by passing a DNA double helix through another, using a transient double-strand break. DNA gyrase catalyzes ATP-dependent negative supercoiling of DNA and is involved in DNA replication, recombination, and transcription. Topoisomerase IV is also involved in supporting DNA replication, and the primary function of this enzyme seems to be the decatenation of multiply linked daughter chromosomes during the terminal stages of DNA replication.It has been proposed that the susceptibility of bacteria to quinolones is determined primarily by which one of the two target enzymes is more sensitive to quinolones (4,6,19). It has been reported that the primary target of many quinolones seems to be topoisomerase IV in Staphylococcus aureus (2-4, 6, 9, 19) and that the primary target in Streptococcus pneumoniae varies among the quinolones (5, 12, 13). Recently, some quinolones were reported to...
Quinolone resistance gene nqr-T91 in a clinical isolate of Pseudomonas aeruginosa P1481 was cotransducible with catA1 in P. aeruginosa PAO. The nqr-T91 transductant, PKH-T91, was resistant to norfloxacin, imipenem, and chloramphenicol and showed less norfloxacin accumulation than the parent strain did. Loss of the 46-kDa outer membrane protein (D2) and an increase in the 50-kDa outer membrane protein in PKH-T91 were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lipopolysaccharides in the transductant were also changed. These alterations were considered to be related to lower levels of norfloxacin accumulation in PKH-T91. These genetic and biochemical properties suggested that an nfxC type of quinoloneresistant mutation occurred in a clinical isolate of P. aeruginosa P1481.New quinolones have potent in vitro antimicrobial activities and are therapeutically effective against gram-positive and gram-negative bacterial infections, including Pseudomonas aeruginosa infection. Recently, quinolone-resistant clinical isolates of P. aeruginosa have emerged because of greater and wider clinical usage of new quinolones (1, 9, 18, 31).Quinolone resistance in bacteria can be attributed to two mechanisms, (i) alteration of DNA gyrase and (ii) alteration of membrane permeability. These mechanisms have also been found in P. aeruginosa. Four chromosomal mutations, nfxA (nalA), nfxB, nfxC, and nalB (cfxB), that confer quinolone resistance have been identified and mapped on the P. aeruginosa PAO chromosome (10,14,28,30). The nfxA and nalA genes are alleles of gyrA that encode DNA gyrase subunit A, while the other genes are associated with the membrane permeability of new quinolones. In clinical isolates of P. aeruginosa, alteration of DNA gyrase or membrane permeability has been reported to be one of the quinolone resistance mechanisms (6,8,17,22,25,29,34).We previously reported on the mechanism of quinolone resistance in a clinical strain of P. aeruginosa P1481 (2) that had been isolated from the urine of a patient treated with norfloxacin in a Japanese hospital and had shown high-level quinolone resistance. The quinolone resistance gene of P1481, nqr-T81, has been reported to be cotransducible with eda (hex)-9001, which is known to be cotransducible with nfxA (gyrA), on the P. aeruginosa PAO chromosome (2). DNA gyrase subunit A purified from P1481 showed resistance to quinolones (2). Thus, quinolone resistance in P1481 was associated with an alteration of DNA gyrase. However, P1481 showed less susceptibility to new quinolones than the gyrA mutant did (2). It was difficult to attribute the high level of quinolone resistance in this strain only to alteration of DNA gyrase. In this study, we examined the quinolone resistance mechanisms in P1481 in addition to alteration of DNA gyrase (gyrA mutation). Table 1 shows the antimicrobial susceptibilities of P1481 and the other strains used in this study. Antimicrobial susceptibility (MIC) was measured by an agar dilution method with MuellerHinton agar (Difco Labora...
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