ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Aug. 1992, p. 1708-1714

Vol. 36, No. 8

0066-4804/92/081708-07$02.00/0 Copyright © 1992, American Society for Microbiology

In Vitro Antibacterial Activity of Q-35, a New Fluoroquinolone TATSUYA ITO,* MASAKO OTSUKI, AND TAKESHI NISHINO

Department of Microbiology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto, 607 Japan

S

Nakauchi-cho,

Received 3 December 1991/Accepted 3 June 1992

The in vitro activity of Q-35, an 8-methoxy fluoroquinolone, was compared with those of ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, and sparfloxacin. The MICs of Q-35 for 90%o of strains tested (MIC90s) of Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes were 0.2, 6.25, 0.2, 0.39, and 0.39 ,ug/ml, respectively. The activity of Q-35 was 4- to 16-fold greater than those of ofloxacin, ciprofloxacin and lomefloxacin but equal to those of tosufloxacin and sparfloxacin against these organisms. For 82 ciprofloxacin-resistant staphylococci (MIC0 = 100 ,ug/ml), Q-35 was the most active of the new quinolones tested (MIC9. = 6.25 p,g/ml). The MIC90s of Q-35 against Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa were 0.2, 0.78, and 12.5 ,ug/ml, respectively, and Q-35 was 2- to 16-fold less active than the other quinolones tested. Q-35 showed potent bactericidal activity and inhibited the supercoiling activity of DNA gyrase of S. aureus, E. coli, and P. aeruginosa. In recent years, some fluoroquinolones with a broad antibacterial spectrum of activity have been developed. However, fluoroquinolone-resistant Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa strains have also appeared (8, 10). In particular, certain methicillin-resistant S. aureus strains have become problematic in clinical settings. Q-35, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3methylamino-piperidine-1-yl)-4-oxoquinoline-3-carboxylic acid (Fig. 1) is a new fluoroquinolone with a broad antibacterial spectrum with improved activity against gram-positive bacteria. Q-35 was first synthesized at Chugai Pharmaceutical Co. Ltd., Shizuoka, Japan. In this paper, we report the in vitro activity of Q-35 in comparison with those of ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, and sparfloxacin against gram-positive and gram-negative clinical isolates. In addition, the bactericidal activity and inhibitory effects of Q-35 on DNA gyrase are reported.

MATERUILS AND METHODS Antimicrobial agents. Q-35 was synthesized as a free base at the Research Foundation of Chugai Pharmaceutical Co. Ltd. Ofloxacin (12) was obtained from Daiichi Pharmaceutical Co. Ltd., ciprofloxacin chloride (2) was obtained from Bayer Pharmaceutical Co. Ltd., tosufloxacin (3, 15) was obtained from Toyama Chemistry Co. Ltd., lomefloxacin (5) was obtained from Hokuriku Pharmaceutical Co. Ltd., and sparfloxacin (7) was obtained from Dainippon Pharmaceutical Co. Ltd. Q-35 and these reference quinolones, except ciprofloxacin chloride, were initially dissolved in 0.1 N NaOH and diluted with water on the day of the assay. Ciprofloxacin chloride was dissolved in water. Bacterial strains. Bacterial strains used in this study were recent clinical isolates collected from various laboratories and hospitals in Japan between 1985 and 1991. All isolates *

Corresponding author. 1708

maintained as frozen (-80°C) stock cultures at Kyoto Pharmaceutical University. Determination of MICs. MICs were determined by the twofold agar dilution method recommended by the Japan Society of Chemotherapy (1), with a sensitivity test (ST) agar (Eiken Chemical Co. Ltd., Tokyo, Japan). The susceptibility of streptococci was determined with ST agar supplemented with 10% sheep blood. The susceptibility of Haemophilus influenzae was determined with ST agar supplemented with 5% Fildes enrichment (GIBCO, Detroit, Mich.). The susceptibility of Moraxella catarrhalis was determined with chocolate ST agar. The inoculated plates were incubated for 24 h at 37°C. The susceptibility of Neissera gonorrhoeae was determined with chocolate ST agar following incubation in a candle jar for 48 h. The overnight broth cultures of the bacterial strains were diluted with broth corresponding to a final concentration of about 106 CFU/ml. Five microliters of each bacterial suspension was applied with an inoculator (Microplanter; Sakura Seisakusho, Tokyo, Japan) onto an agar plate containing twofold serial dilutions of quinolones, delivering a final inoculum of 5 x 103 CFU per spot. The MIC was defined as the lowest concentration of the compound that prevented visible growth on the agar. Bactericidal activity. The bactericidal activity of Q-35 against S. aureus Smith, Escherichia coli KC-14, and P. aeruginosa E-2 was determined. Mid-logarithmic-phase cells (approximately 106 CFU/ml, viable count) were exposed to quinolones at concentrations of 1/4 x, 1/2 x, 1 x, 2 x, and 4 x MIC in ST broth (Eiken Chemical Co. Ltd.). One-hundredmicroliter samples were removed at fixed times, and several dilutions were prepared in saline as needed and plated onto ST agar. Drug carryover was reduced by 100-fold dilution of the sample with agar. The number of colonies were counted after 24 h of incubation at 37°C. Inhibition of DNA gyrase. (i) Isolation of E. coli and P. aeruginosa DNA gyrase. E. coli NIHJ JC-2 and P. aeruginosa PAO-1 were used as the source of DNA gyrase. Crude enzyme was prepared from cells following treatment with were

IN VITRO

VOL. 36, 1992

FIG. 1. Chemical structure of Q-35.

lysozyme (Sigma), streptomycin, and ammonium sulfate, as previously described (11). The crude enzyme was loaded on a novobiocin-Sepharose (Pharmacia, Tokyo, Japan) column (13) followed by a stepwise gradient with 0.2 M KCI, 2 M KCl, and 2 M KCl-5 M urea in TED buffer (50 mM Tris hydrochloride [pH 7.5], 1 mM EDTA, 0.5 mM dithiothreitol). The gyrase holoenzyme of E. coli was eluted with 2 M KCl-5 M urea. The A subunit of P. aeruginosa was eluted with 0.2 M KCl. The active fraction was further purified by using a heparin-Sepharose CL-6B (Pharmacia) column. Active fractions were eluted around 0.2 M NaCl. The B subunit of P. aeruginosa was eluted with 2 M KCl-5 M urea from a novobiocin-Sepharose column. (ii) Isolation of S. aureus DNA gyrase. S. aureus FDA 209P was used as the source of DNA gyrase. Crude enzyme was prepared by the method of Okuda et al. (9), with slight modification. Phenylmethylsulfonyl fluoride was used as protease inhibitor in the crude cell extract. The crude enzyme was loaded onto a novobiocin-Sepharose column and was eluted stepwise with 0.1 M NaCl, 2 M NaCl, and 2 M NaCl-5 M urea in TGED buffer (50 mM Tris hydrochloride [pH 8.0], 1 mM EDTA, 10% glycerol, 1 mM dithiothreitol). The A subunit was eluted with 2 M NaCl. The B subunit was eluted with 2 M NaCl-5 M urea. (iii) DNA supercoiling assay and IC50 determinations. The supercoiling activity of DNA gyrase and 50% inhibitory concentration (IC50) of quinolones were examined by using modifications of methods described in previous reports (9, 11, 14). One unit of gyrase as defined by Gellert et al. (4) is the amount of enzyme that catalyzes the conversion of one-half of the relaxed pBR322 DNA to the supercoiled form in 1 h at 37°C in the assay mixture containing 0.1 ,ug of DNA. Approximately 2 U of gyrase holoenzyme of E. coli was used per reaction. Approximately 2 U each of gyrase A and B subunits of P. aeruginosa and S. aureus was used per reaction. The reaction mixture containing A and B subunits, drug solution, and relaxed pBR322 was incubated at 37°C for 1 h. The reaction was stopped by the addition of sodium dodecyl sulfate at a final concentration of 1.3% and subjected to 0.8% agarose gel electrophoresis. The gel was stained with ethidium bromide (2 ,ug/ml) and photographed with a UV transilluminator. The negatives were traced with a densitometer. RESULTS

Antibacterial activity. The in vitro activities of Q-35, ofloxacin, ciprofloxacin, tosufloxacin, lomefloxacin, and sparfloxacin against gram-positive and gram-negative bacteria are shown in Table 1. The MIC of Q-35 for 90% of the strains (MIC90) of methicillin-susceptible S. aureus was 0.2 ,ug/ml, which was

ACTIVITY

OF Q-35

1709

4- to 16-fold lower than those of ofloxacin, ciprofloxacin, and lomefloxacin but equal to or 2-fold higher than those of sparfloxacin and tosufloxacin. For methicillin-resistant S. aureus the MIC90 of Q-35 was 6.25 ,g/ml and was the lowest of the quinolones studied. The MIC90 of Q-35 for S. epidermidis was 0.2 ,ug/ml, which was 8- to 16-fold lower than those of ofloxacin, ciprofloxacin, and lomefloxacin but equal to those of tosufloxacin and sparfloxacin. Eighty-two strains (25.1%) of the staphylococci tested were ciprofloxacin resistant (MIC > 3.13 ,ug/ml) (6). The MIC90 of Q-35 for these strains was 6.25 ,ug/ml and was the lowest of the quinolones tested. The MIC90 of Q-35 for both Streptococcus pneumoniae and Streptococcus pyogenes was 0.39 p,g/ml, which was lower than those for ofloxacin, ciprofloxacin, and lomefloxacin but equal to, or higher than, those for tosufloxacin and sparfloxacin. The MIC90 of Q-35 was 0.78 for Enterococcus faecalis, 1.56 for Enterococcus faecium, and 0.78 ,ug/ml for Enterococcus avium, Q-35 being more potent than ofloxacin and lomefloxacin but equal in potency to ciprofloxacin, tosufloxacin, and sparfloxacin. The MIC90s of Q-35 for E. coli, Citrobacter freundii, Kiebsiella pneumoniae, Enterobacter cloacae, Enterobacter aerogenes, Proteus vulgaris, and Morganella morganii were 100 0.025->100 0.78->100 0.05-25

0.2 0.78 1.56 0.05 3.13 0.1

6.25 50 50 25 100 12.5

Q-35

0.012-1.56 0.1-25 0.012-25 0.025-6.25 0.05-100 0.05-12.5

0.1 0.39 0.2 0.05 0.78

0.2 1.56 1.56 0.2 3.13 0.2

0.012-0.78 0.1-1.56 0.012-1.56 0.012-0.39 0.05-12.5 0.025-3.13

0.1 0.39 0.39 0.05 0.78 0.1

0.2

Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin Sparfloxacin

Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin Sparfloxacin

Ciprofloxacin-susceptiblec staphylococci (245)

Ciprofloxacin-resistant staphylococci (82)

S. pneumoniae (33)

Q-35

Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin Sparfloxacin

1.56 12.5 12.5 1.56 25 3.13

6.25 50 100 50 100 12.5

Q-35

0.1-6.25 0.39-100 0.2-50 0.05-12.5 0.39-100 0.1-50

0.39 1.56 1.56 0.2 6.25 0.39

0.39 3.13 3.13 0.39 12.5 0.39

Q-35

0.1-0.39 0.78-1.56 0.39-0.78 0.1-0.2 1.56-6.25 0.2-0.78

0.2 0.78 0.39 0.1 3.13 0.2

0.39 1.56 0.78 0.2 6.25 0.39

0.2-0.78

0.78 3.13 1.56 0.39 6.25 0.78 1.56 3.13 1.56

Sparfloxacin Q-35

Tosufloxacin Lomefloxacin

3.13-12.5

Sparfloxacin

0.2-0.78

0.39 1.56 0.78 0.39 6.25 0.39

0.2-3.13 0.78-12.5 0.2-12.5

0.78 3.13 0.78

Ofloxacin

Ciprofloxacin

E. faecium (60)

0.78 0.78 0.1 1.56 0.1

0.2-25 0.78-100 3.13->100 0.05->100 3.13->100 0.05-25

Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin

E. faecalis (40)

0.1

90%

Q-35 Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin Sparfloxacin

Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin Sparfloxacin S. pyogenes (34)

MIC (Wg/ml)a

Drug

Q-35 Ofloxacin

Ciprofloxacin

0.78-3.13 0.2-1.56

0.1-0.78

Continued on following page

IN VITRO ACTIVITY OF Q-35

VOL. 36, 1992

1711

TABLE 1-Continued

Organism

Drug

(no. of strains)

0.78 6.25 0.78

0.1-1.56 0.39-6.25 0.1-3.13 0.025-0.78 0.78-12.5 0.1-1.56

0.39 1.56 0.39 0.2 6.25 0.39

0.78 3.13 1.56 0.39 12.5 0.78

0.1-0.78 0.05-0.78 0.012-0.2 0.025-0.2 0.1-1.56 0.012-0.39

0.2 0.1 0.025 0.025 0.2 0.025

0.2 0.1 0.025 0.05 0.2 0.05

0.1-6.25 0.05-3.13 0.012-0.39 0.025-1.56 0.2-3.13 0.025-3.13

0.78 0.2 0.05 0.2 0.39 0.2

1.56 0.39 0.2 0.39 0.78 0.78

0.1-3.13 0.05-1.56 0.025-0.39 0.012-0.39 0.1-1.56 0.025-0.78

0.39 0.1 0.05 0.05 0.2 0.1

1.56 0.78 0.2 0.2 1.56 0.39

0.2-6.25 0.05-0.78 0.012-0.39 0.025-0.78 0.1-1.56 0.025-3.13

0.39 0.1 0.025 0.05 0.2 0.05

0.78 0.39 0.1 0.1 0.39 0.2

Q-35

0.2-3.13 0.05-0.78 100 0.2-> 100 0.05-50

6.25 3.13 0.78 0.39 6.25 1.56

100 50 100 25 50 12.5

S. marcescens (39)

Q-35 Ofloxacin Ciprofloxacin Tosufloxacin Lomefloxacin Sparfloxacin

0.39-100 0.1-100 0.05-25 0.05->100 0.2-50 0.05-50

6.25 1.56 0.78 0.78 3.13 1.56

50 25 12.5 12.5 25 12.5

P. aeruginosa (50)

Q-35

1.56-50 0.78-12.5 0.1-0.78 0.1-1.56 1.56-6.25 0.39-3.13

6.25 1.56 0.39 0.39 1.56 1.56

12.5 3.13 0.78 0.78 6.25 3.13

Q-35

0.2-6.25 0.1-3.13 0.1-3.13 0.025-0.39 0.39-12.5 0.012-0.78

0.39 0.2 0.2 0.05 0.78 0.05

0.78 0.78 0.78 0.1 1.56 0.1

Q-35

0.2-0.025 0.025-0.2