In conjugational crosses, three Klebsiella pneumoniae strains and one Serratia marcescens strain have been demonstrated to transfer resistance determinants to newer types of cephalosporins. While Klebsiella strains donated cefotaxime, cefamandole and cefuroxime resistance to Escherichia coli K-12 recipients, the genetic analysis of exconjugants after the transfer of plasmids from Serratia strains to Proteus or Salmonella recipients showed that the cefoxitin resistance determinant was also co-transferred. In subsequent transfer cycles of this plasmid, cefotaxime and cefoxitin resistance determinants segregated in contrast to the relative stability of plasmids derived from Klebsiella strains in subsequent transfer cycles. From results obtained in this study, it may be concluded that in some strains of nosocomial Enterobacteriaceae, resistance to newer cephalosporins could be transmissible and thus plasmid-located.

We isolated an imipenem-resistant strain, GN17203, of Pseudomonas aeruginosa. The strain produced a I8-lactamase that hydrolyzed imipenem. The I-lactamase was encoded by a 31-MDa plasmid, pMS350, which belongs to incompatibility group P-9. The plasmid conferred resistance to f8-lactams, gentamicin, and sulfonamide and was transferable by conjugation to P. aeruginosa but not to Escherichia coli. The molecular weight of the purified enzyme was estimated to be 28,000, and the isoelectric point was 9.0. The enzyme showed a broad substrate profile, hydrolyzing imipenem, oxyiminocephalosporins, 7-methoxycephalosporins, and penicillins. The enzyme activity was inhibited by EDTA, iodine, p-chloromercuribenzoate, CuS04, and HgCl2 but not by clavulanic acid or sulbactam.

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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...

Two genetically distinct classes of norfloxacin-resistant Pseudomonas aeruginosa PA04009 mutants were isolated spontaneously. Two norfloxacin resistance genes, nfxA and nfxB, were mapped between hex-9001 and leu-9005 and between pro-9031 and ilv-9023, respectively, on the P. aeruginosa PAO chromosome. The nfxA gene was shown to be an allele of nalA by transductional analysis with bacteriophage F116L. The nfxB mutant showed a 16-fold increase in resistance to norfloxacin and a slight increase in resistance to nalidixic acid. The nfxB mutant was unique in that it showed hypersusceptibility to beta-lactam and aminoglycoside antibiotics. This mutant had about a threefold-lower rate of norfloxacin uptake than that of the wild-type strain or nfxA mutant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins demonstrated the appearance of a 54,000-dalton protein in the nfxB mutant. These findings suggested that the norfloxacin resistance mechanism in the nfxB mutant might be an alteration in outer membrane permeability to norfloxacin.Norfloxacin and other new 4-quinolones show potent antibacterial activity against gram-negative and grampositive bacteria (11,14,20). They also have high in vitro and in vivo antibacterial activity against Pseudomonas aeruginosa strains that show a strong intrinsic resistance to various antimicrobial agents including older quinolones such as nalidixic acid (11,20,42). The high antibacterial activity of new quinolones might be due to their strong inhibitory action against DNA gyrase, a target enzyme of quinolones, which has been isolated from various bacteria including Pseudomonas aeruginosa (5,16,26,28,42).Drug resistance mediated by plasmids or transposons is a serious clinical problem. 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). 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). Two loci coding for resistance to nalidixic acid, nalA and nalB, have also been mapped on the P. aeruginosa PAO chromosome (32). It has been reported that DNA replication is resistant to nalidixic acid in permeabilized cells of nalA mutants and that nalB mutants cause a decrease of cell permeability to nalidixic acid and carbenicillin (32).To gain information on the resistance mechanisms to norfloxacin in P. aeruginosa, we isolated spontaneous...