ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1986, p. 248-253 Vol. 30, No. 2 0066-4804/86/080248-06$02.00/0 Copyright © 1986, American Society for Microbiology Isolation and Characterization of Norfloxacin-Resistant Mutants of Escherichia coli K-12 KEIJI HIRAI,'* HIROSHI AOYAMA,1 SEIGO SUZUE,1 TSUTOMU IRIKURA,1 SHIZUKO IYOBE,2 AND SUSUMU MITSUHASHI3 Central Research Laboratories, Kyorin Pharmaceutical Co. Ltd., Nogi-machi, Shimotsuga-gun, Tochigi,l Department of Microbiology, School ofMedicine, Gunma University, Maebashi, Gunma,2 and Episome Institute, Fujimi, Gunma,3 Japan Received 30 December 1985/Accepted 19 May 1986 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 hydropho- bicity 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 activ- lated from E. coli KL-16 by plating on nutrient agar plates ity against gram-positive and gram-negative bacteria, includ- containing norfloxacin. Strain KJC-1, a gIpT derivative of ing Pseudomonas aeruginosa, have been developed recently JC1552, was made by fosfomycin selection as described (12, 18, 33, 34). They also show high antibacterial activity previously (21). The phages Tula and TuIb were kindly against nalidixic acid-resistant strains including mutational provided by S. Mizushima of Nagoya University, Nagoya, resistant strains such as gyrA and nalB mutants of Esche- Japan. richia coli K-12 (14, 28). There is incomplete cross- Drugs. AM-833, ciprofloxacin, norfloxacin, ofloxacin, resistance between the new quinolones and nalidixic acid oxolinic acid, pipemidic acid, piromidic acid, and rosoxacin (14, 17, 28, 35). were synthesized by Central Research Laboratories of The high antibacterial activity of new quinolones (e.g., Kyorin Pharmaceutical Co., Ltd. Ampicillin, cefoxitin, norfloxacin and ciprofloxacin) might be due to their strong chloramphenicol, cloxacillin, fosfomycin, gentamicin, nali- inhibitory action on DNA gyrase, which is the target enzyme dixic acid, novobiocin, streptomycin, and tetracycline were of quinolones (K. Sato, Y. Inoue, T. Fujii, H. Aoyama, M. obtained from commercial sources. Inoue, and S. Mitsuhashi, personal communication). Re- Determination of hydrophobicity of quinolones. Hydropho- cently, we found that the bacterial outer membrane penetra- bicity of quinolones was determined by a previously de- tion mechanisms of new quinolones differed from those of scribed method (13). Solutions (10 ,ug/ml) of quinolones were old quinolones such as nalidixic and piromidic acids (13). made in 0.1 M phosphate buffer (pH 7.2). After shaking with To study the mechanisms of resistance to norfloxacin, the an equal volume of n-octanol at 25°C for 48 h and centrifug- first-developed new quinolone, spontaneous norfloxacin- ing at 1,870 x g for phase separation, the concentrations of resistant mutants were isolated from E. coli K-12 and their quinolones in the aqueous phase were determined by a properties were investigated. Three classes of mutants spectrophotometric assay. The partition coefficients (P) showed less susceptibility to norfloxacin; one class appeared were expressed as the ratio of the amount of compound in to consist of gyrA mutants, while the other two were novel the n-octanol phase to that in the aqueous phase. mutants that showed alteration in norfloxacin uptake. In this Media. Antibiotic medium 3, Mueller-Hinton medium, report we describe the biochemical and genetic properties of Mueller-Hinton agar, and nutrient agar were purchased from these new mutants in detail. Difco Laboratories, Detroit, Mich. L-broth and L-agar were (This work was presented in part at the 14th International prepared as described previously (22). Minimal medium Congress of Chemotherapy, Kyoto, Japan, 23-28 June (citrate-free minimal A medium) (6) contained 0.5% sugar 1985.) (glucose, galactose, or lactose) and was supplemented with amino acid (1 mM), if necessary. MATERIALS AND METHODS Measurement of susceptibility to antimicrobial agents and other chemical agents. MICs were determined by the agar Bacterial strains and phages. Bacterial strains and phages dilution method (18). An overnight culture of the bacterial used in this study are listed in Table 1. Spontaneous norflox- strain in Mueller-Hinton broth was diluted 100-fold with acin-resistant mutants (KEA12, KEA13, KEA16) were iso- fresh broth, and 5 ,ul of the bacterial suspension (about 5 x 106 cells per ml) was inoculated with an inoculator * Corresponding author. (Microplanter; Sakuma Seisakusho, Tokyo, Japan) onto 248 VOL. 30, 1986 NORFLOXACIN-RESISTANT MUTANTS OF E. COLI K-12 249 TABLE 1. Bacterial strains and phages used in this study KE7 (OmpC deficient) and strain KEll (OmpF deficient), E. coli K-12 which have been described previously (13). derivative or Genotype or phenotype Source Uptake of norfloxacin by the bacterial cells. The uptake of bacteriophage norfloxacin by bacterial cells was measured by a method described (13). Cells were grown to mid-log phase E. coli K-12 previously KL-16 Hfr relA thi B. Bachmann at 37°C in antibiotic medium 3, and the bacterial cell suspen- KEA12 Norfloxacin-resistant mutant This study sion (A570 = 0.7) was prepared with the same medium. of KL-16 Norfloxacin was added to the bacterial suspensions to a final KEA13 Norfloxacin-resistant mutant This study concentration of 10 ,ug/ml, and the cultures were incubated of KL-16 at 37°C with shaking. After the indicated times, 10 ml of the KEA16 Norfloxacin-resistant mutant This study culture was chilled, and the cells were sedimented by of KL-16 centrifugation and washed once in 2 ml of saline. The cells JC1552 F- argG gal his lacY leu B. Bachmann were then suspended in 1 ml of saline. The suspension was malA metB mtl strA supE immersed in boiling water for 7 min and then centrifuged. tonA trp tsx xyl KJC-1 glpT derivative of JC-1552 This study The concentration of norfloxacin in the supernatants was AB3505 F- argH galK his ilvD lacY B. Bachmann measured by a bioassay with E. coli NIHJ JC-2. or X malA metE mtl proA trp tsx xyl RESULTS LC607 F- ara lac leu lys metE K. Matsubara Isolation of norfloxacin-resistant mutants. Spontaneous proC purE str thi trp xyl mutants were isolated from E. coli Bacteriophages norfloxacin-resistant T2, T3, T4, This laboratory KL-16 by plating approximately 1010 CFUs of a late- T5, T7, P1 exponential-phase culture on nutrient agar plates containing Tula OmpF-specific phage S. Mizushima norfloxacin. Mutants showing less susceptibility to norflox- TuIb OmpC-specific phage S. Mizushima acin could be obtained at frequencies of 10-9 to 10-10 by selection on nutrient agar plates containing 0.05, 0.1, or 0.2 p,g of norfloxacin per ml. Mutants resistant to higher con- Mueller-Hinton agar plates containing serial twofold dilu- centrations of norfloxacin (0.39 pug/ml in nutrient agar) could tions of the agents. MICs were determined after 18 h of not be obtained by spontaneous single-step mutations. Mu- incubation at 37°C. tants with low-level resistance fell into three types according Genetic analysis. Conjugation, growth of bacteriophages, to their susceptibility to norfloxacin and nalidixic acid. Three and transduction were carried out as described by Miller representative mutants (KEA12, KEA13, KEA16) were (23). The procedure for interrupted mating was as described chosen for further study. by Curtiss (5). Acquisition of glpT+ after transduction was Susceptibility to quinolones and other chemical agents. The selected for by growth on minimal agar plates supplemented MIC was determined for quinolones (Table 2) and other with 0.4% L-a-glycerolphosphate as the sole carbon source. agents, including various antibiotics (Table 3). Strain KEA12 Phage sensitivity test. Phage susceptibility was determined showed especially high resistance to nalidixic and piromidic by a spot test (11). Bacteria (approximately 2 x 108 cells) acids. The MICs of norfloxacin and other new quinolones were mixed with 3 ml of L-broth containing 0.7% (wt/vol) such as ciprofloxacin, ofloxacin, and AM-833 for KEA12 agar (45°C) and poured over the surface of an L-agar plate. were increased eightfold over those of KL-16. Mutant After 20 min, bacteriophage suspensions (about 1010 PFU) KEA12 showed no changes in susceptibility to other antimi- were spotted onto the plate with an inoculator. The plates crobial agents, dyes, or detergents. Strain KEA13 showed were then incubated at 37°C for 24 h prior to scoring. low-level resistance to all the quinolones that were tested. Hydrophobicity of bacterial cell surface. Bacterial cell This mutant also showed a slight but reproducible increase in surface hydrophobicity was measured by a modification of resistance to ampicillin, chloramphenicol, cloxacillin, the method of Rosenberg et al. (24). The bacteria were novobiocin, and tetracycline and a larger increase in resist- grown at 37°C with shaking in antibiotic medium 3.