Isolation and characterization of norfloxacin-resistant mutants of Escherichia coli K-12

Abstract

We isolated spontaneous mutants from Escherichia coli K-12 with low-level resistance to norfloxacin. These mutants were classified into the following three types on the basis of their properties: (i) NorA appeared to result for mutation in the gyrA locus for the A subunit of DNA gyrase; (ii) NorB showed low-level resistance to quinolones and other antimicrobial agents (e.g., cefoxitin, chloramphenicol, and tetracycline), and the norB gene was considered to map at about 34 min on the E. coli K-12 chromosome; (iii) NorC was less susceptible to norfloxacin and ciprofloxacin but was hypersusceptible to hydrophobic quinolones such as nalidixic acid and rosoxacin, hydrophobic antibiotics, dyes, and detergents. Susceptibility to bacteriophages and the hydrophobicity of the NorC cell surface also differed from that of the parent strain. The norC gene was located near the lac locus at 8 min on the E. coli K-12 chromosome. Both NorB and NorC mutants had a lower rate of norfloxacin uptake, and it was found that the NorB mutant was altered in OmpF porin and that the NorC mutant was altered in both OmpF porin and apparently in the lipopolysaccharide structure of the outer membrane.Many new quinolones showing potent antibacterial activity against gram-positive and gram-negative bacteria, including Pseudomonas aeruginosa, have been developed recently (12,18,33,34). They also show high antibacterial activity against nalidixic acid-resistant strains including mutational resistant strains such as gyrA and nalB mutants of Escherichia coli K-12 (14, 28). There is incomplete crossresistance between the new quinolones and nalidixic acid (14,17,28,35).The high antibacterial activity of new quinolones (e.g., norfloxacin and ciprofloxacin) might be due to their strong inhibitory action on DNA gyrase, which is the target enzyme of quinolones (K. Sato, Y. Inoue, T. Fujii, H. Aoyama, M. Inoue, and S. Mitsuhashi, personal communication). Recently, we found that the bacterial outer membrane penetration mechanisms of new quinolones differed from those of old quinolones such as nalidixic and piromidic acids (13).To study the mechanisms of resistance to norfloxacin, the first-developed new quinolone, spontaneous norfloxacinresistant mutants were isolated from E. coli K-12 and their properties were investigated. Three classes of mutants showed less susceptibility to norfloxacin; one class appeared to consist of gyrA mutants, while the other two were novel mutants that showed alteration in norfloxacin uptake. In this report we describe the biochemical and genetic properties of these new mutants in detail.( lated from E. coli KL-16 by plating on nutrient agar plates containing norfloxacin. Strain KJC-1, a gIpT derivative of JC1552, was made by fosfomycin selection as described previously (21). The phages Tula and TuIb were kindly provided by S. Mizushima of Nagoya University, Nagoya, Japan.Drugs. AM-833, ciprofloxacin, norfloxacin, ofloxacin, oxolinic acid, pipemidic acid, piromidic acid, and rosoxacin were synthesized by Central Research Laborato...

Mutations producing resistance to norfloxacin in Pseudomonas aeruginosa

“…Antibiotic medium 3, Mueller-Hinton agar, Mueller-Hinton broth, Proteose Peptone, and nutrient agar were purchased from Difco Laboratories, Detroit, Mich. L broth, L agar, and minimal medium (citrate-free minimal A medium) were as described previously (13,30). Minimal A medium contained either 0.4% glucose or 20 mM pyruvate as a carbon source and was supplemented with 1 mM amino acids and purine, if necessary.…”

“…We previously found (13) OmpF. The outer membrane proteins of the nfxB mutant (KH4013E), the nfxA mutant (KH4023), and the wild-type strain (PA04009) were prepared by the method of Poxton et al (31) and were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis.…”

“…Several chromosomal mutations, gyrA, nalB, nalC (gyrB), and nalD, conferring nalidixic acid resistance were identified and mapped on the Escherichia coli K-12 chromosome (10,17,18). Recently, we (13) and Hooper et al (16) identified norfloxacin resistance mutations (norA, norB, norC, nfxA, and nfxB) in E. coli K-12. norA and nfxA were alleles of gyrA encoding the A subunit of DNA gyrase, while norB, norC, and nfxB determined outer membrane permeability resistance to norfloxacin, were associated with a decrease in OmpF porin protein, and were mapped at 34 min, near 8 min, and at 20 to 22 min, respectively (13,16).…”

“…Recently, we (13) and Hooper et al (16) identified norfloxacin resistance mutations (norA, norB, norC, nfxA, and nfxB) in E. coli K-12. norA and nfxA were alleles of gyrA encoding the A subunit of DNA gyrase, while norB, norC, and nfxB determined outer membrane permeability resistance to norfloxacin, were associated with a decrease in OmpF porin protein, and were mapped at 34 min, near 8 min, and at 20 to 22 min, respectively (13,16). Two loci coding for resistance to nalidixic acid, nalA and nalB, have also been mapped on the P. aeruginosa PAO chromosome (32).…”

“…However, resistance to nalidixic acid and other quinolones in bacteria is due to chromosomal mutations (10,13,(16)(17)(18)32). Plasmids or transposons that carry quinolone resistance genes have not been found in bacteria (4).…”