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...

The intracellular antimicrobial activity of tosufloxacin was tested against SalmoneUla enteritidis C-32 by using human lung fibroid WI-38 cells and was compared with those of ofloxacin and norfloxacin. The intracellular antimicrobial activities of these drugs were evaluated by determining the numbers of viable organisms remaining within cells after treatment with various drug concentrations. At 0.2 and 0.78 ,ug/ml, tosufloxacin suppressed intracellular multiplication of S. enteritidis C-32 more effectively than ofloxacin and norfloxacin did. The ability of tosufloxacin to penetrate into WI-38 cells was also determined by the velocity gradient method. The ratio of the intracellular concentration to the extracellular concentration of tosufloxacin was 1.7-and 2.6-fold higher than those of ofloxacin and norfloxacin, respectively. The results indicate that the potent intracellular bactericidal activity of tosufloxacin may be due not only to its high in vitro activity but alsQ to its ability to penetrate into cells at a high level.It is known that intracellular parasites, such as members of the genera Salmonella (3), Shigella (19), Legionella (7,15,17), and Staphylococcus (13,18), invade host cells and grow. Therefore, these pathogens evade the bactericidal potential of antimicrobial agents such as ,-lactams that do not enter host cells (7,8). The survivability of these pathogens in host cells makes drug therapy difficult.Tosufloxacin, 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 newly developed quinolone antimicrobial agent and has shown a broad spectrum of activity against grampositive and gram-negative bacteria (4, 5). Tosufloxacin has potent in vitro antibacterial activity against intracellular parasites, including the genera Salmonella and Shigella. Tosufloxacin also has a high level of efficacy against intestinal infections caused by Salmonella species in clinical trials in Japan (G. Masuda, H. Sagara, R. Nakaya, T. Yasuda, and T. Noumi, Program Abstr. 28th Intersci. Conf. Antimicrob. Agents Chemother., abstr. no. 251, 1988). In order to clarify in detail the reason for the good clinical response of tosufloxacin, the intracellular activity of tosufloxacin against Salmonella species and its ability to penetrate into normal human cells were examined by using tissue culture cells of the human lung fibroid cell line WI-38 and were compared with those of ofloxacin and norfloxacin. MATERIALS AND METHODSBacterial strain and susceptibility test. Salmonella enteritidis C-32 was isolated from a patient with intestinal infection and was stored at -120°C until use. The susceptibility of this organism to drugs was ascertained by determining the MIC by the broth dilution method. Eagle growth medium (Nissui Seiyaku, Tokyo, Japan) containing 10% fetal bovine serum (GIBCO Laboratories, Grand Island, N.Y.) was used for the assay medium. The MIC was determined after incubation for 18 h at 370C. * Correspondi...