T-3262, a new fluoroquinolone, showed a broad spectrum of activity against gram-positive and gramnegative bacteria. T-3262 had most potent activity against gram-positive cocci, such as Staphylococcus, Streptococcus, and Enterococcus spp. The MICs of T-3262 for 90% of strains tested were between 0.05 and 1.56 ,ug/ml. Against members of the family Enterobacteriaceae and Pseudomonas aeruginosa, the activities of T-3262 were almost equal to those of ciprofloxacin. Obligate anaerobes were also susceptible to T-3262. T-3262 was bactericidal for one strain each of Staphylococcus aureus, Escherichia coli, and P. aeruginosa at concentrations near its MIC; and fluoroquinolones, including T-3262, inhibited DNA gyrase activity at low concentrations. The 50% effective dose of T-3262 after oral administration against systemic infections with S. aureus in mice was about 6 times lower than that of ofloxacin and about 20 times lower than that of norfloxacin.Nalidixic acid (2) displays relatively good activity against gram-negative bacteria and is a useful agent in the treatment of urinary tract infections. However, against gram-positive bacteria, its activity is limited, as are those of oxolinic acid (20), cinoxacin (12), and pipemidic acid (16). Over the quarter of a century since the discovery of nalidixic acid, many chemical modifications to the 4-quinolone nucleus have been attempted to get more useful compounds. The 6-fluoro-substituted-4-quinolone nucleus expanded the spectrum and enhanced antimicrobial activities. Norfloxacin (10), ofloxacin (15), ciprofloxacin (21), enoxacin (11), AM-833 (7), and belong to the fluoroquinolones.T-3262, the p-toluenesulfonic acid salt of DL-7-(3-amino-1 -pyrrolidinyl) -1 -(2,4-difluorophenyl)-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-carboxylic acid monohydrate, is a new fluoroquinolone (Fig. 1) which possesses excellent activity against gram-positive and gram-negative bacteria. We compared its in vitro and in vivo antibacterial activities with those of ciprofloxacin, ofloxacin, norfloxacin, and pipemidic acid.MATERIALS AND METHODS Antimicrobial agents. T-3262 was synthesized at the Research Laboratories, Toyama Chemical Co., Ltd., Toyama, Japan; ciprofloxacin was from Bayer Yakuhin, Osaka, Japan; ofloxacin and nalidixic acid were from Daiichi Seiyaku Co., Ltd., Tokyo, Japan; norfloxacin was from Kyorin Seiyaku, Tokyo, Japan; pipemidic acid was from Dainippon Seiyaku Co., Ltd., Osaka, Japan; gentamicin was from Shionogi Seiyaku Co., Ltd., Osaka, Japan; methicillin was from Banyu Seiyaku Co., Ltd., Tokyo, Japan. All the quinolones were dissolved in 0.1 N NaOH and diluted in distilled water appropriately.Test strains. Clinical isolates were acquired from the Laboratory of Drug Resistance in Bacteria, Gunma University, Gunma, Japan. Staphylococcus aureus Smith, Escherichia coli ML4707, and Pseudomonas aeruginosa GN11189 * Corresponding author.were used for time kill curves, determination of frequency of spontaneous mutants, and systemic infection in mice. E. coli ML4707 and P. aerugi...

DNA gyrase is a bacterial enzyme which catalyzes the ATP-dependent negative supercoiling of DNA. It is the accepted target of quinolones. The enzyme from Citrobacter freundii 1ID976 was purified by affinity chromatography on novobiocin-Sepharose and heparin-Sepharose. It had two subunits, designated A and B, which closely resembled those of the enzyme from Escherichia coli and Micrococcus luteus in enzymatic requirements. The inhibitory effects of the quinolones on the supercoiling activities of the enzyme correlated with their antibacterial activities. New quinolones were better inhibitors of DNA gyrase than nalidixic acid and pipemidic acid. We also purified DNA gyrase from a spontaneous nalidixic acid-resistant mutant (M2-5). The gyrases from IID976 and M2-5 were defined as mixtures of subunits As+Bs (s, susceptible) and Ar+Br (r, resistant), respectively. The supercoiling activities of reconstituted Ar+Br and Ar+Bs were more resistant to quinolones than As+Bs and As+Br. These findings indicate that one mechanism of C. freundii resistance against quinolones is resistance modification of the A subunit protein.An essential enzyme in Escherichia coli, termed DNA gyrase, was discovered by Gellert et al. (8). This enzyme has been shown to have several activities in vitro, including the introduction of negative superhelical twists into covalently closed circular DNA and the reversible interlocking of duplex DNA circles like links in a chain. In vivo, DNA gyrase is required for DNA replication and for transcription from certain operons in E. coli, and it is involved in DNA repair and recombination. Since these discoveries were made with the DNA gyrase of E. coli, analogous gyrases have been found in Micrococcus luteus (22), Bacillus subtilis (27, 37), and Pseudomonas aeruginosa (23).The enzyme is composed of two subunits. The A subunit protein mediates the ability of the enzyme to introduce and rejoin double-strand breaks in DNA. The structurally related antimicrobial agents nalidixic acid, oxolinic acid, and their analogs, including new quinolone compounds, are inhibitors of the A subunit (7,17,32). The B subunit protein mediates energy transduction and ATP hydrolysis and is inhibited by novobiocin, coumermycin Al, clorobiocin, and their analogs (14,24,38). Nalidixic acid is a particularly active agent against most enteric gram-negative bacteria, but not against P. aeruginosa and gram-positive bacteria. Addition of a 7-piperazinyl and a 6-fluorine to the nalidixic acid molecule has resulted in a number of compounds termed the new quinolones, such as norfloxacin (18), ofloxacin (31), ciprofloxacin (25), and AM-833 (12) with potent broad-spectrum antibacterial activity, including activity against P. aeruginosa, gram-positive bacteria, and most nalidixic acid-resistant strains. Furthermore, previous reports showed that the new quinrolones inhibited DNA gyrase activity at a lower concentration than nalidixic acid and pipemidic acid (4, 32).Drug resistance mediated by plasmids or transposons is a serious clinical prob...

About 30% of 150 recent clinical isolates of Citrobacterfreundii were resistant to .3.13 ,Ig of norfloxacin and ofloxacin per ml. Study of one quinolone-resistant strain for which the norfloxacin MIC was 100 ,Ig/ml suggested that resistance was associated with both an altered A subunit of DNA gyrase and reduction in drug uptake accompanied by a decrease in an outer membrane protein.

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The uptakes of norfloxacin by quinolone-resistant and -susceptible strains of Serratia marcescens were almost the same and 50% inhibitory concentrations for DNA gyrase and the MICs of quinolones were correlated, suggesting that DNA gyrase alterations are the basis of quinolone resistance.Quinolones are having increasing clinical use because of their potent and broad antimicrobial activity. Accompanying this usage, the number of quinolone-resistant strains has increased (4, 13,14,21). Especially in Serratia marcescens, there are many strains resistant to quinolones (6). Moreover, this organism has easily acquired and showed resistance to many antibiotics (10, 15) and has been recognized as an important nosocomial pathogen. However, only preliminary investigation (5) has been reported concerning DNA gyrase (8) in this organism. We report here the purification of DNA gyrase from S. marcescens, the inhibitory eflects of quinolones on DNA gyrase supercoiling activity, and norfloxacin uptake into bacterial cells.S. marcescens IAM1184 was a stock collection strain of the Institute of Applied Microbiology, University of Tokyo. S. marcescens GN16707 and GN16720 were isolated from urinary tract infections.T-3262, ofloxacin, norfloxacin, and pipemidic acid were commercially available materials. Susceptibility to quinolones was measured by agar dilution using Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.) with an inoculum of approximately 104 CFU per spot. Crude extract of DNA gyrase was prepared as previously described (1), applied to a novobiocin-Sepharose (19) column (1.0 by 10 cm), and eluted in steps with 0.1 M KCl, 2 M KCl, and 5 M urea in TED (10 mM Tris hydrochloride [pH 7.5], 1 mM EDTA [pH 7.5], 0.5 mM dithiothreitol) bufler. Active fractions eluting with 2 M KCl were loaded onto a heparin-Sepharose CL-6B column (1.0 by 15 cm), and the activity was eluted with 0.5 M KCI. The fraction eluting with 5 M urea was again purified on novobiocin-Sepharose. Active fractions were pooled and dialyzed against 50% glycerol in TED bufler. The assay of DNA gyrase was slightly modified from previous reports (7). Reaction mixtures for DNA supercoiling activity contained 20 mM Tris hydrochloride (pH 7.5), 40 mM KCl, 1 mM MgCt2, 1 mM spermidine, 1 mM dithiothreitol, 10 ,ug of bovine serum albumin per ml, 30 .zg of tRNA per ml, 1.5 mM ATP, 0.1 ,ug of pBR322 DNA which had been relaxed by topoisomerase I, two active proteins (1 U each), and drug solution. One unit of DNA gyrase was defined as the activity that converted 50% of relaxed pBR322 DNA to the supercoiled form. We measured 50% inhibitory doses (IC50s) as previously described (17). The uptake of norfloxacin by S. marcescens was measured by bioassay as described previ- (20).To determine whether the two proteins were actually components of S. marcescens DNA gyrase, we assayed their supercoiling activities. Neither protein by itself had any activity. However, supercoiling activity was observed when the two proteins were combined. Moreover, we tested the supercoiling act...