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. The ance to cefoxitin, but it showed no increase in resistance to bacteria were harvested at early log phase, washed twice, aminoglycoside antibiotics. The third mutant (KEA16) was and suspended in PUM buffer (pH 7.1), which consisted of unique in that it showed a slight but reproducible increase the following: 22.2 g of K2HPO4 * 3H20, 7.26 g of KH2PO4, (generally two- to fourfold) in resistance to hydrophilic 1.8 g of urea, 0.2 g of MgSO4 * 7H20 and distilled water to 1,000 ml. p-Xylene (2 ml) was added to 5 ml of bacterial cell TABLE 2. Susceptibility of mutants to quinolones suspension (A570 = 1.0) in PUM buffer. Following 10 min of preincubation at 37°C, the mixtures were agitated uniformly MIC (p.g/ml)b for the following with a Vortex mixer for 120 s. After a 15-min period during Compound Hydrophobicity mutants: which the p-xylene phase rose completely, the aqueous KL-16 KEA12 KEA13 KEA16 was absorbance was deter- phase removed, and the light Norfloxacin 0.01 0.05 0.39 0.20 0.20 mined at 570 nm. Cell-surface hydrophobicity was expressed AM-833 0.08 0.05 0.39 0.20 0.05 as the ratio of decrease in absorbance of the aqueous Ciprofloxacin 0.02 0.025 0.20 0.10 0.05 phase. Nalidixic acid 3.92 3.13 100 12.5 0.78 Characterization of outer membrane proteins. Outer mem- Ofloxacin 0.33 0.05 0.39 0.20 0.05 brane proteins were prepared by the method of Sawai et al. Oxolinic acid 2.23 0.20 6.25 1.56 0.10 (27) and analyzed by sodium dodecyl sulfate-polyacrylamide Pipemidic acid 0.03 1.56 25 6.25 3.13 gel electrophoresis as described by Uemura and Mizushima Piromidic acid 11.7 12.5 400 50 1.56 (32). Gels were stained with Coomassie brilliant blue. To Rosoxacin 10.7 0.10 6.25 0.78 0.0125 identify OmpF and OmpC porin proteins, we used as a a Partition coefficient (P) in n-octanol-0.1 M phosphate buffer (pH 7.2). reference the outer membrane proteins prepared from strain b MIC determined by the agar dilution method. 250 HIRAI ET AL. ANTIMICROB. AGENTS CHEMOTHER.

TABLE 3. Susceptibility of mutants to antibiotics and TABLE 4. Conjugational mapping of the norC mutant other agents Coinheritance of resistancea (%) of the Antibiotics, MIC (p.g/ml) for the following mutants: Selected following recipients: detergents, marker and dyes KL-16 KEA12 KEA13 KEA16 JC1552 AB3505 LC607 Ampicillin 6.25 6.25 12.5 3.13 Trp+ (27)" <1 (0/87)c <1 (0/100) Cefoxitin 3.13 3.13 25 6.25 Gal' (17) < 1 (0/87) < 1 (0/100) Chloramphenicol 6.25 6.25 12.5 3.13 PurE+ (12) 30 (40/134) Cloxacillin 400 400 800 12.5 PurC + (9) 78 (114/146) Gentamicin 1.56 1.56 1.56 1.56 Lac+ (8) 89 (136/152) 69 (62/90) 93 (91/98) Novobiocin 400 400 800 0.39 ProA+ (6) 47 (33/71) Streptomycin 12.5 12.5 12.5 12.5 Leu+ (2) 49 (49/101) Sodium dodecyl >12,800 >12,800 >12,800 200 a Norfloxacin (0.1 ,ug/ml) resistance. sulfate b Numbers in parentheses are map positions (in minutes). Gentian violet 25 25 25 0.39 c Numbers in parentheses are number of Norr transconjugants per number of transconjugants tested. quinolones such as norfloxacin, ciprofloxacin, and pipemidic type of mutant (KEA13) with JC1552. Transfer of the norB acid but was hypersusceptible to hydrophobic quinolones gene appeared at about 10 min after his and at about 7 min such as nalidixic acid, rosoxacin, and piromidic acid. It was before trp; the resistance gene therefore was considered to also less susceptible to cefoxitin but showed hypersuscept- be at about 34 min on the map of E. coli K-12. Uninterrupted ibility to cloxacillin, novobiocin, sodium dodecyl sulfate, mating with LC607, AB3505, and JC1552 suggested that the and gentian violet. norC gene (in KEA16) was located near lac (Table 4). All of Mapping of resistance genes. It was shown by genetic the norfloxacin-resistant transconjugants tested had the mapping that the resistance genes of these mutants are same pattern of susceptibility to quinolones and other chem- located at distinct chromosomal positions. The resistance ical agents as the donor strain (KEA16). However, it was not genes of KEA12, KEA13, and KEA16 were designated as possible to analyze the position of norC by P1 transduction norA, norB, and norC, respectively. In interrupted mating because this mutant was resistant to P1 phage (see below). with JC1552, the norA gene (in KEA12) entered about 4 min Phage susceptibility and hydrophobicity of the cell surface before his and would, therefore, be at about 48 min on the of mutants. The susceptibility of mutants to chemical agents chromosome map of E. coli K-12, where gyrA is located (9). suggested that KEA13 and KEA16 might be altered in outer It was also demonstrated that norA was cotransducible with membrane properties. Because alterations of the outer mem- gipT at frequencies of about 50% by P1 transduction (data brane have been related to changes in bacteriophage suscep- not shown). These results suggest that KEA12 is due to a tibility, we investigated the susceptibility of these mutants to mutation in the gyrA locus for subunit A of DNA gyrase. In various phages, including phages Tula and TuIb (Table 5). Fig. 1 are shown the kinetics of gene transfer in the second The norA mutant was susceptible to all bacteriophages tested, as was the parent strain. On the other hand, KEA13 was resistant to Tula phage and KEA16 was resistant to 600 - phages Tula, P1, and T4. It is well known that Tula and TuIb use the OmpF and OmpC porin proteins, respectively, as His' parts of their receptors (19) and that P1 and T4 phages use the lipopolysaccharide of the outer membrane as a receptor (4, 29). The phage susceptibility test indicated that KEA13 might be altered in OmpF porin proteins and that KEA16 might be altered in both OmpF porin proteins and the 400 NFLr lipopolysaccharide of the outer membrane. The cell surface of KEA16 also became more hydrophobic than that of the ci parent strain (Table 5).

0 TrP* Analysis of outer membrane proteins of mutants. Outer c membrane proteins of mutants were prepared and separated

E by sodium dodecyl sulfate-polyacrylamide gel electrophore- 200 sis. In Fig. 2 it is demonstrated that OmpF proteins were significantly reduced in KEA13 and KEA16 mutants in

TABLE 5. Phage susceptibility and surface hydrophobicity of mutants Phage susceptibility of the following phagesa: Hydro- Strain phobicity 0 10 20 30 40 50 60 T2 T3 T4 'E7 P1 Tula TuIb (pb) Time after mating (min) KL-16 S S S S S S S <0.01 KEA12 S S S S S S S <0.01 FIG. 1. Marker entry time for a mating between KEA13 and KEA13 S S S S S R S <0.01 JC1552. The donor was counterselected with streptomycin (200 KEA16 S S PR S R R S 0.37 ,ug/ml). Norfloxacin (0.1 ,ug/ml) was present in selective plating media for norfloxacin resistance. Abbreviations: His, histidine; Trp, a S, Susceptible; PR, partially resistant; R, resistant. tryptophan; NFLXr, norfloxacin resistance. b p, Partition coefficient (p-xylene-PUM buffer). VOL. 30, 1986 NORFLOXACIN-RESISTANT MUTANTS OF E. COLI K-12 251

the parent strain, although these mutants remained suscep- Omp C tible to a concentration of 0.39 ,ug/ml or less. The gyrA (norA) mutant that showed a high level of resistance to nalidixic acid (MIC, 100 pug/ml) remained relatively suscep- Omp F ftow tible to new quinolones. Sato et al. (personal communica- Omp A u tion) have demonstrated that new quinolones such as nor- floxacin and ciprofloxacin inhibit DNA gyrase isolated from E. coli KL-16 at significantly lower concentrations than does nalidixic acid. It is considered likely that new quinolones also retain considerable inhibitory activity against DNA gyrase of the gyrA mutant (KEA12). Quinolone-resistant mutants of Klebsiella pneumoniae showing alterations in outer membrane proteins were also reported by Sanders et al. (25) and Gutmann et al. (8). In this study, mutants lacking OmpF proteins were found among spontaneous, single-step, norfloxacin-resistant mutants of E. coli K-12. Our result that a norfloxacin-resistant mutation was associated with de- creased outer membrane protein OmpF coincided with the 1 2 3 results of the study by Hooper et al. (15). Mutants lacking FIG. 2. Sodium dodecyl sulfate-polyacrylamide gel electropho- OmpF protein easily can be obtained spontaneously by resis of outer membrane proteins of strain KL-16 (lane 1), KEA13 selection of beta-lactam antibiotic-resistant or chloramphen- (lane 2), and KEA16 (lane 3). Approximately 25 ,ug of protein was icol-resistant mutants of E. coli (10, 19, 20), and the mutants loaded onto the gel. demonstrate increased resistance to antibiotics passing through this porin. We found previously that quinolones comparison with the parent strain. It appeared that OmpC penetrated the outer membrane of E. coli K-12 through the proteins were also slightly reduced in the KEA16 mutant. OmpF porin (13); those findings were confirmed in this The phage susceptibility of these mutants was confirmed by study. this electrophoretic analysis of outer membrane proteins. It was known that the structural gene (ompF) and regula- Uptake of norfloxacin by bacterial cells. It has been re- tory genes (ompB locus, including ompR and envZ) of the ported that the mechanism of resistance to nalidixic acid in OmpF porin protein were located at 21 and 75 min, respec- E. coli is an alteration in subunits of DNA gyrase and tively, on the E. coli K-12 chromosome (1, 26, 30). Hooper permeability through the bacterial cell membrane (3, 8, 16, et al. (15) found that the nfxB norfloxacin-resistant mutation 17, 28, 35). To confirm whether an alteration of permeability that was associated with additional resistances to tetracy- of norfloxacin into bacterial cells could be the mechanism of cline, chloramphenicol, and cefoxitin and with decreased resistance in norB and norC mutants, we measured their OmpF porin protein also mapped between 20 and 22 min. In uptake of norfloxacin and compared it with that of the parent contrast, our norB mutant lacking the OmpF protein mapped strain (Fig. 3). Mutants KEA13 and KEA16 showed about a at about 34 min. It was reported previously (7) that amplifi- twofold lower norfloxacin uptake than the wild-type strain able resistance to tetracycline, chloramphenicol, and other (KL-16), suggesting that alterations in the OmpF protein of unrelated antimicrobial agents, including nalidixic acid, was the outer membrane lead to decreased permeability of nor- mediated by the marA (for multiple antibiotic resistance) floxacin through the outer membrane of E. coli K-12. locus and that this locus mapped at 34 min on the E. coli K-12 chromosome. Expression of phenotypes of marA was DISCUSSION temperature sensitive, but a norB mutant could express its phenotypes at 42°C (data not shown). However, the map There has been interest in resistance mechanisms to new quinolones that show high antibacterial activity against gram-positive and gram-negative bacteria, including nali- dixic acid-resistant strains. It has been reported that resist- >> 0.3 ance mechanisms to nalidixic acid in E. coli are alterations in subunits A and B of DNA gyrase and penetration of the drug through the bacterial cell membrane (3, 16, 17, 28). Several E studies of norfloxacin resistance mechanisms have been 0.2 - reported (2, 25, 31); however, mechanisms of resistance to a new quinolones such as norfloxacin and ciprofloxacin have I LL not been studied in detail until quite recently. Recently, z Hooper et al. (15) reported that norfloxacin-resistant mu- -+- 0.1 tants obtained by serial passage on an agar plate with an 0 increasing norfloxacin concentration showed alterations in the DNA gyrase A subunit and in the OmpF outer membrane porin protein. :D ,-, In this study we also demonstrated changes in three distinct loci that resulted in less susceptibility to norfloxacin Incubation Time (min) as a result of an alteration in DNA gyrase or in the outer FIG. 3. Uptake of norfloxacin (NFLX) by mutants of E. coli membrane porin protein. These three types of mutants were KL-16. Symbols: 0, KL-16 (wild type); 0, KEA13 (norB); A, less susceptible to norfloxacin and ciprofloxacin than was KEA16 (norC). 252 HIRAI ET AL. ANTIMICROB. AGENTS CHEMOTHER. position and multiple resistance phenotype of marA are ACKNOWLEDGMENTS considerably similar to those of norB; therefore, it would be We thank K. Sato and co-workers for communicating the details interesting to clarify the correlation between marA and norB of their work on DNA gyrase prior to publication and S. Mizushima genes. This should be tested in further studies. At present for providing TuIa and TuIb phages. We also thank Hiroko the function of this gene (norB) is unclear, but it may Kawahara and Toshiya Ikeda for preparation of the manuscript. regulate OmpF protein production. There is a possibility that norB, like nfxB (15), is a previously unidentified locus LITERATURE CITED affecting ompF expression. 1. Bachmann, B. J., and K. B. Low. 1983. Linkage map of Escherichia coli K-12. Microbiol. Rev. 47:180-230. The norC mutant showed the same susceptibility to hy- 2. Barry, A. L., and R. N. Jones. 1984. Cross-resistance among drophobic quinolones and antibiotics as the lipopolysaccha- cinoxacin, ciprofloxacin, DJ-6783, enoxacin, nalidixic acid, nor- ride-deficient (rfaE) mutant of Salmonella typhimurium (13). floxacin, and oxolinic acid after in vitro selection of resistant Furthermore, resistance to phages P1 and T4 and cell surface populations. Antimicrob. Agents Chemother. 25:775-777. hydrophobicity changes suggest that the norC mutant may 3. Bourguignon, G. L., M. Levitt, and R. Sternglanz. 1973. Studies be of the rough (heptose-deficient) type. The norC mutant on the mechanism of action of nalidixic acid. Antimicrob. was also altered in OmpF proteins, as evidenced by the loss Agents Chemother. 4:479-486. of susceptibility to TuIa and analysis of outer membrane 4. Colemann, W. G., and L. Leive. 1979. Two mutations which affect the barrier function of the Escherichia coli K-12 outer proteins by sodium dodecyl sulfate-gel electrophoresis. De- membrane. J. Bacteriol. 139:899-910. fects have been observed in the composition of outer mem- 5. Curtiss, R., III. 1981. Gene transfer, p. 243-265. In P. Gerhardt, brane proteins of heptose-deficient mutants of E. coli K-12 R. G. E. Murray, R. N. Costilow, E. W. Nester, W. A. Wood, (11); therefore, an alteration in the OmpF protein of the norC N. R. Krieg, and G. B. Phillips (ed.), Manual of methods for mutant might be closely correlated with a defect in its general bacteriology. American Society for Microbiology, lipopolysaccharide core. The norC mutation was located Washington, D.C. near lac (8 min) on the E. coli chromosome. The acrA and 6. Davis, B. D., and E. S. Mingioli. 1950. Mutants of Escherichia lpcA mutants, showing hypersusceptibility to hydrophobic coli requiring methionine or vitamin B12. J. Bacteriol. 60:17-28. antibiotics, dyes, and detergents, are located at 11 and 6 min, 7. George, A. M., and S. B. Levy. 1983. Gene in the major respectively, on the E. coli chromosome (1, 4). The cotransduction gap of the Escherichia coli K-12 linkage map pheno- required for the expression of chromosomal resistance to tetra- type of the acrA mutant closely resembles that of the norC cycline and other antibiotics. J. Bacteriol. 155:541-548. mutant, but the former has normal levels and types of outer 8. Gutmann, L., R. Williamson, N. Moreau, M.-D. Kitzis, E. membrane proteins (4). The lpcA (6 min) mutation is likely to Collatz, J. F. Acar, and F. W. Goldstein. 1985. Cross-resistance be genetically distinct from the norC mutation because a to nalidixic acid, trimethoprim, and chloramphenicol associated selected lac (8 min) locus coinherits norC with higher with alterations in outer membrane proteins of Klebsiella, frequency than does a selected proA (6 min) locus, and Enterobacter, and Serratia. J. Infect. Dis. 151:501-507. susceptibility of these mutants to T3 and T7 phages is also 9. Hane, M. W., and T. H. Wood. 1969. Escherichia coli K-12 distinct (11). But, the results presented in this report, how- mutants resistant to nalidixic acid: genetic mapping and domi- nance studies. J. Bacteriol. 99:238-241. ever, do not exclude the possibility that the norC gene is an 10. Harder, K. J., H. Nikaido, and M. Matsuhashi. 1981. Mutants of allele of lpcA or acrA. Escherichia coli that are resistant to certain beta-lactam com- We found previously (13) that hydrophobic quinolones pounds lack the OmpF porin. Antimicrob. Agents Chemother. such as nalidixic acid might be able to penetrate through the 20:549-552. lipid bilayer and that lipopolysaccharide-deficient mutants of 11. Havekes, L. M., B. J. J. Lugtenberg, and W. P. M. Hoekstra. S. typhimurium show hypersusceptibility to these com- 1976. Conjugation deficient E. coli K-12 F- mutants with pounds. The norC mutant is hypersusceptible to hydropho- heptose-less lipopolysaccharide. Mol. Gen. Genet. 146:43-50. bic quinolones, suggesting a defect in the outer membrane 12. Hirai, K., H. Aoyama, M. Hosaka, Y. Oomori, Y. Niwata, S. permeability barrier to these agents or exposure of the Suzue, and T. Irikura. 1986. In vitro and in vivo antibacterial activity of AM-833, a new quinolone derivative. Antimicrob. hydrophobic region on the outer membrane. The low-level Agents Chemother. 29:1059-1066. resistance to hydrophilic quinolones such as norfloxacin in 13. Hirai, K., H. Aoyama, T. Irikura, S. lyobe, and S. Mitsuhashi. KEA16 might be due to a decrease in OmpF porin protein 1986. Differences in susceptibilities to quinolones of outer and not to changes in lipopolysaccharide. Results of this membrane mutants of Salmonella typhimurium and Escherichia study emphasize our previous finding (13) that the outer coli. Antimicrob. Agents Chemother. 29:535-538. membrane permeability of new quinolones differs from that 14. Hirai, K., A. Ito, S. Suzue, T. Irikura, M. Inoue, and S. of old quinolones such as nalidixic and piromidic acids. This Mitsuhashi. 1982. Mode of action of AM-715, a new nalidixic might be one of the reasons for incomplete cross-resistance acid analog. Gunma Reports on Med. Sci. 19:375-392. 15. Hooper, D. C., J. S. Wolfson, K. S. Souza, C. Tung, G. L. among quinolones. McHugh, and M. N. Swartz. 1986. 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Furthermore, the norB and norC 14:240-245. 18. Ito, A, K. Hirai, M. Inoue, H. Koga, S. Suzue, T. Irikura, and S. mutants lacking OmpF porin show a lower growth rate than Mitsuhashi. 1980. In vitro antibacterial activity of AM-715, a the gyrA mutant and the wild-type strain. In particular, norC new nalidixic acid analog. Antimicrob. Agents Chemother. mutant showed hypersusceptibility to animal serum (data 17:103-108. not shown). These results suggest that emergence of norB 19. Jaffe, A., Y. A. Chabbert, and E. Derlot. 1983. Selection and and norC type mutants is unlikely to occur in vivo. characterization of,3-lactam-resistant Escherichia coli K-12 VOL. 30, 1986 NORFLOXACIN-RESISTANT MUTANTS OF E. COLI K-12 253

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