ANTIMICROBLAL AGENTS AND CHEMOTHERAPY, Oct. 1992, p. 2108-2117 0066-4804/92/102108-10$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 36, No. 10

In Vitro and In Vivo Antibacterial Activities of AM-1155, a New 6-Fluoro-8-Methoxy Quinolone MASAKI HOSAKA, TOKUTARO YASUE, HIDEYUKI FUKUDA, HIROSHI TOMIZAWA, HIROSHI AOYAMA, AND KEIJI HIRA*

Central Research Laboratones, Kyorin Phannaceutical Co., Ltd., Nogi-machi, Shimotsuga-gun, Tochigi, 329-01, Japan Received 6 January 1992/Accepted 17 July 1992

AM-1155 is a new quinolone with a wide spectrum of antibacterial activity against various bacteria including anaerobes and Mycoplusma pneumoniae. AM-1155 was 2- to 16.fold more active than ciprofloxacin and ofloxacin against Staphylococcus aureus including methicillin-resistant strains, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis; its MICs for 90%o of strains tested were 0.10 to 0.78 ,g/ml. The activity of AM-1155 was comparable to that of ciprofloxacin against members of the family Enterobacteriaceae, Branhamella catr*als Haemophilus influenzae, and Neisseria gonorrhoeae, but was fourfold less than that of ciprofloxacin against Pseudomonas aerugios. Against Xanthomonas maltophiia, Acinetobacter cales, and Campylobacter jejuni, AM-1155 was two- to fourfold more active than ciprofloxacin. At a concentration of 1.56 ,ig/ml, AM-1155 inhibited 901% of Bdcteroidesfragilis strains tested; its activity was 8- to 10-fold higher than those of ofloxacin and ciprofloxacin. Development of resistance to AM-1155 in S. aureus and S. epidermidis occurred at a lower frequency than did that to ciprofloxacin after eight transfers in the presence of drug. In the oral treatment of mouse systemic infections, AM-1155 was four- to eightfold more effective than ciprofloxacin against gram-positive cocci and was as active as ciprofloxacin against gram-negative rods. The efficacy of an oral or a subcutaneous dose of AM-llSS was two- to fivefold greater than that of ofloxacin. Against experimental pneumonia with Kiebsiella pneumoniae and P. aeruguiosa, AM-1155 was two- to fourfold more active than ciprofloxacin and ofloxacin. AM-1155 also had good efficacy against mouse ascending urinary tract infections with Escherichia coli and P. aeruginosa. These results suggest that AM-llSS may be a potent antibacterial agent applicable to various infections. ,

A number of new quinolones with a broad spectrum of activity against gram-positive and gram-negative bacteria have been developed during the past decade; and some compounds, such as norfloxacin (12), ofloxacin (17), and ciprofloxacin (18), have been introduced into clinical practice and have proven to be efficacious in the therapy of bacterial infections. Nevertheless, these new quinolones show only moderate activity against gram-positive and anaerobic bacteria. AM-1155, 1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy7-(3-methyl-1-piperazinyl)-4-oxo-3-quinolinecarboxylic acid, is a new quinolone that has been developed by Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan (Fig. 1). In the study described here, we compared the in vitro and in vivo antibacterial activities of AM-1155 with those of ciprofloxacin, norfloxacin, and ofloxacin. (This work was presented in part at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy

[91.)

MATERIALS AND METHODS Antibacterial agents. AM-1155, ciprofloxacin, norfloxacin, and ofloxacin were synthesized at the Central Research Laboratories of Kyorin Pharmaceutical Co., Ltd. Nalidixic acid was obtained from Sigma Chemical Co., St. Louis, Mo. Organisms. The organisms used in this study were laboratory strains and clinical isolates which were collected between 1986 and 1990 from several hospitals in Japan. The *

Corresponding author.

organisms were kept as stock cultures in 10% skim milk at -40°C in our laboratory. Quinolone-resistant mutants of Escherichia coil and Pseudomonas aeruginosa-E. coli KEA12 (8), E. coli KEA13 (8), E. coli KE7 (7), P. aeruginosa KH4023 (10), P. aenrginosa KH4013E (10), and P. aeruginosa KH4014a (2)-were used for the study on susceptibility and inhibition of DNA gyrase supercoiling activity. E. coli MH5 and N-51 were provided by M. Inoue (Gunma University, Gunma, Japan) and S. Nakamura (Dainippon Pharmaceutical Co., Ltd., Osaka, Japan), respectively. Determination of MICs. The MICs of the compounds were determined by the twofold agar dilution method (6) with Mueller-Hinton medium (Difco), which was supplemented with 5% defibrinated horse blood for streptococci, enterococci, and Campylobacter jejuni. Chocolate agar was used for Haemophilus influenzae and Neisseria spp. GAM agar (Nissui Seiyaku Co., Tokyo, Japan) and buffered starchyeast extract agar were used for obligate anaerobes and Legionella pneumophila, respectively. The inocula were prepared as follows. All strains except streptococci, enterococci, H. influenzae, Neisseria spp., L. pneumophila, and Bacteroidesffragilis were grown overnight in Mueller-Hinton broth (Difco). Streptococci and enterococci were grown overnight in Mueller-Hinton broth supplemented with 5% defibrinated horse blood. H. influenzae and B. fragilis were grown in brain heart infusion broth plus 5% Fildes extract (Kanto Kagaku Co., Tokyo, Japan) and GAM broth (Nissui Seiyaku Co.), respectively. Neisseria spp. and L. pneumophila were grown overnight on chocolate agar or buffered starch-yeast extract agar plates. Bacterial colonies were removed just before use and were suspended in saline. The 2108

ANTIBACTERIAL ACTIVITY OF AM-1155

VOL. 36, 1992 0

#H33 FIG. 1. Chemical structure of AM-1155.

overnight broth cultures and bacterial suspensions of Neisseria spp. and L. pneumophila were diluted in fresh broth to a final concentration of approximately 106 CFU/ml, and final inoculum, 5 x 103 CFU per loopful) one loopful (5 of each bacterial suspension was applied onto the agar plates with an inoculater (Microplanter; Sakuma Seisakusho, Tokyo, Japan). The plates were incubated at 37°C for

pl;

18 h; plates containing C jejuni and L. pneumophila incubated for 48 h. Neisseria spp., H. influenzae, and C. jejuni were incubated in the GasPak system (BBL Microbiology Systems), and obligate anaerobes were incubated in an anaerobic chamber (Forma Scientific). The drug susceptibility of Mycoplasma pneumoniae was determined by the twofold broth dilution method described previously (13). The MIC was defined as the lowest concentration of antimicrobial agent that inhibited visible growth. Determination of MBCs. The MBCs for 10 strains each of Staphylococcus aureus, E. coli, and P. aeruginosa were determined by the method of Pearson et al. (15). Before determination of MBCs, the MICs were determined by the twofold broth dilution method with Mueller-Hinton broth. For the MIC determination, the final inoculum ranged from 2 x 105 to 2 x 106 CFU/ml. The MBCs were determined by transferring 50 ,ul of culture broth from each MIC tube without visible growth onto drug-free Mueller-Hinton medium plates. After 18 h of incubation at 37°C, the number of bacterial colonies occurring on the plates was determined. The MBC was defined as the lowest concentration of drug which killed >99.9% of the inoculum. Time-kill study. The bactericidal activity of AM-1155 was measured by the method described previously (6). Representative organisms incubated in Mueller-Hinton broth (Difco) for 18 h at 37°C were diluted with fresh broth to about 105 CFU/ml, and the diluted cultures were incubated for 2 h at 37°C with shaking. After 2 h of preincubation, AM-1155 was added to the culture at various concentrations around the MIC. Portions (0.1 ml) of the cultures were removed at the indicated times and were plated onto drug-free MuellerHinton medium (Difco) plates, after dilution as necessary. The number of colonies were counted after 18 h of incubation at 37°C. Inhibition of DNA gyrase. Bacterial DNA gyrase was purified from E. coli KL16 (5.9 x 10' U/mg of protein), E. coli N-51 (gyrA) (2.5 x 105 U/mg of protein), P. aeruginosa PA01840 (4.4 x 104 U/mg of protein), and P. aeruginosa PKH-T477 (gyrA) (4.9 x 104 U/mg protein) as described previously (3, 4, 11, 16). The DNA gyrase of Micrococcus luteus ATCC 4698 (100 U/13 pl) was obtained from a commercial source (Bethesda Research Laboratories, Gaithersburg, Md.). The assay of inhibition of DNA supercoiling

were

2109

activity of DNA gyrase was performed as described by Gellert et al. (5). Relaxed plasmid pBR322 substrate DNA was prepared by treatment with calf thymus topoisomerase I as recommended by the manufacturer (Bethesda Research Laboratories). One unit of gyrase activity was defined as the amount of enzyme that catalyzed the conversion of 50% of the relaxed closed-circular pBR322 DNA to the supercoiled form in 2 h at 37°C in a gyrase reaction mixture. Mutation frequency of resistance. The test organisms were grown in Mueller-Hinton broth at 37°C with shaking until the midexponential growth phase was achieved. The bacteria were then concentrated by centrifugation, and approximately 108 to 109 CFU of bacteria was plated onto MuellerHinton medium plates containing each test compound at the concentration of four times the corresponding MIC. Colonies were counted after 48 h of incubation at 37°C. The frequency of spontaneous mutations selected by each compound was calculated as the ratio of the number of cells growing on drug-containing agar plates to the number of inoculated cells. Development of resistance. AM-1155 and ciprofloxacin were prepared as twofold dilutions in Mueller-Hinton broth. The test strains were inoculated into a dilution series of each agent with an inoculum of 105 CFU/ml. After overnight incubation at 37°C, the broth with the highest concentration of the drug showing turbidity in each series was subcultured into a new dilution series of the same agent. This was done for 8 consecutive days. Mouse protection test. Male ICR mice weighing about 20 g (SLC Japan Inc., Shizuoka, Japan) were infected intraperitoneally with bacterial suspensions. S. aureus and P. aeruginosa were injected as suspensions in 5% gastric mucin (Difco). The other strains were used as suspensions in saline. The test compounds were administered orally or subcutaneously 1 h after challenge. Untreated and treated groups at each dose were composed of 10 mice each. The 50% effective doses (ED50s) were calculated by the probit (1) method on the basis of the number of survivors at 7 days after infection. Experimental pneumonia in mice. Experimental pneumonia in mice was induced by Kiebsiella pneumoniae B54 and P. aeruginosa S51 as described previously (6). Male ICR mice weighing about 20 g (SLC Japan Inc.) were placed in an exposure chamber (Ikemoto Rika, Tokyo, Japan) and were infected by exposure to aerosol which was produced by nebulization of 10 ml of the bacterial suspension (1.1 x 109 CFU/ml) at a pressure of 1 kg/cm2 for 30 min. For P. aeruginosa pneumonia, we used leukopenic mice, which were prepared by intraperitoneal injection of cyclophosphamide (250 mg/kg of body weight) at 4 days before infection. The test compounds were administered orally to groups of five mice each at 18, 42, and 66 h after K pneumoniae infection or 8 h after P. aeruginosa infection. The ED50s were calculated by the probit method from the number of survivors at 6 and 7 days after infection for K pneumoniae and P. aeruginosa, respectively. Pharmacokinetics in mice. The tissue distribution and urinary excretion of AM-1155 and ciprofloxacin in male ICR mice weighing 24 to 34 g (SLC Japan Inc. and Clea Japan Inc., Tokyo, Japan) were determined following administration of a single oral dose of 10 mg/kg. At 0.5, 1, 2, 4, 6 and 8 h after administration, blood and tissue samples such as lung and kidney were collected from five mice. Serum was obtained from blood by centrifugation at 1,870 x g for 20 min. Tissue samples were homogenized in 1/15 M phosphate buffer (pH 7.4). After centrifugation, the super-

2110

ANTrMICROB. AGENTS CHEMOTHER.

HOSAKA ET AL.

TABLE 1. Comparative in vitro activities of AM-1155, ciprofloxacin, norfloxacin, and ofloxacin against clinical isolatese

Organism (no. of isolates)

DrugRange

MIC (pg/ml) 5

90%

Staphylococcus aureus (34)

AM-1155 CPFX NFILX OFLX

0.05-0.20 0.20-0.78 0.39-3.13 0.20-0.78

0.10 0.39 1.56 0.39

0.10 0.78 3.13 0.39

Methicillin-resistant Staphylococcus aureus (30)

AM-1155 CPFX NFLX OFLX

0.05-0.20 0.39-3.13 1.56-25 0.39-1.56

0.10 0.78 1.56 0.39

0.20 3.13 12.5 1.56

NFLX- and CPFX-resistant Staphylococcus aureus (10)

AM-1155 CPFX NFLX OFLX

3.13 50 100 25

6.25 100 100 50

Staphylococcus epidemnidis (26)

AM-1155 CPFX NFLX OFLX

1.56-6.25 12.5-100 50-100 6.25-50 0.05-0.39 0.20-1.56 0.39-12.5 0.20-1.56

0.10 0.39 0.78 0.39

0.20 0.39 1.56 0.78

NFLX- and CPFX-resistant Staphylococcus epidennidi (26)

AM-1155 CPFX NFLX

0.20-50 1.56-100 12.5-100 1.56-100

1.56 50 100 12.5

OFLX Staphylococcus haemolyticus (25)

AM-1155 CPFX NFLX

OFLX Staphylococcus hominis (15)

AM-1155 CPFX NFLX

OFLX

Staphylococcus saprophyticus (8)

AM-llSS CPFX NFLX OFLX

Steptococcus pneumoniae (15)

AM-1155 CPFX NFLX OFLX

Streptococcus pyogenes (6)

AM-l155 CPFX

NFL:X OFLX Enterococcusfaecalis (14)

AM-l155 CPFX NFLX

OFLLX Enterococcusfaecium (12)

Escherichia coli (26)

AM-l155 CPFX NFX OFLX AM-l155 CPFX

Klebsiella pneumoniae (24)

NFLX OFLX AM-l155 CPFX NFLX OFLX

50 100 100 100

0.10-6.25 0.20-100 0.39-> 100 0.20-50

0.20 1.56 12.5 1.56

3.13 12.5 100 12.5

0.10-12.5 0.10->100 0.39->100 0.20-100

0.20 0.39 0.78 0.39

6.25 100 >100 50

0.20-6.25 0.20-100 0.78->100 0.39-50 0.20-0.39 0.39-1.56 3.13-12.5 1.56-3.13

0.39 0.78 6.25 1.56

0.39 1.56 12.5 3.13

0.39 0.39-0.78 1.56-3.13 1.56

0.39-0.78 0.39-3.13 3.13-25 1.56-6.25

0.39-3.13 0.78-6.25 6.25-25 3.13-12.5 0.0125-0.05 0.0063-0.05 0.05-0.10

0.05-0.20 0.05-0.78 0.025-0.39 0.10-1.56

0.10-1.56

0.78 1.56 25 6.25 0.78 1.56 3.13 1.56 12.5 6.25 12.5 6.25 0.05 0.025 0.025 0.025 0.10 0.10 0.10 0.10 0.39 0.05 0.39 0.05 0.20 1.56 1.56 0.20 Continued on foUowing page 0.39 0.78 3.13 1.56

ANTIBACTERIAL ACTIVITY OF AM-1155

VOL. 36, 1992

2111

TABLE 1-Continued Organism (no. of isolates)

Drug Range

Enterobacter cloacae (25)

AM-1155 CPFX NFLX OFLX

0.025-0.20 0.0125-0.10 0.05-0.78 0.05-0.39

Serratia marcescens (25)

AM-llSS

0.39-6.25 0.10-3.13 0.20-12.5 0.39-12.5

CPFX NFLX OFLX

MIC (,ug/ml) 50%

90%

0.05 0.025 0.20 0.10

0.05 0.05 0.39 0.20

0.78 0.78 3.13 1.56

1.56 1.56 12.5 6.25

Citrobacterfreundii (12)

AM-1155 CPFX NFLX OFLX

0.05-1.56 0.0125-1.56 0.05-6.25 0.10-6.25

0.05 0.025 0.05 0.10

0.78 0.20 1.56 1.56

Proteus vulganis (11)

AM-1155 CPFX NFLX OFLX

0.10-0.39 0.025-0.10 0.05-0.39 0.10-0.39

0.20 0.025 0.05 0.10

0.20 0.10 0.20 0.20

Proteus mirabilis (10)

AM-1155 CPFX NFLX OFLX

0.20-0.39 0.025-0.10 0.05-0.39 0.10-0.39

0.20 0.025 0.05 0.20

0.20 0.05 0.10 0.20

Morganella morganii (13)

AM-1155 CPFX NFLX OFLX

0.05-0.20 0.0125-0.025 0.025-0.05 0.05-0.20

0.10 0.0125 0.05 0.10

0.10 0.025 0.05 0.10

AM-1155 CPFX

0.10-100 0.0125->100 0.05->100 0.10->100

1.56 6.25 25 6.25

6.25 12.5 100 25

1.56 0.39 0.78 3.13

3.13 0.78 3.13 6.25

Providencia

rettgeri

(12)

NFLX OFLX

Pseudomonas aeruginosa (35)

AM-1155 CPFX NFLX OFLX

0.78-12.5 0.10-1.56 0.39-6.25 0.78-12.5

NFLX- and CPFX-resistant Pseudomonas aeruginosa (34)

AM-1155 CPFX NFLX OFLX

6.25-> 100 3.13-100 12.5->100 12.5-> 100

Xanthomonas maltophilia (13)

AM-1155 CPFX NFLX OFLX

0.20-3.13 0.78-6.25 6.25-25 0.78-6.25

0.78 3.13 12.5 1.56

3.13 6.25 25 6.25

Acinetobacter calcoaceticus (17)

AM-1155 CPFX

0.025-0.10 0.10-0.39 0.78-3.13 0.20-0.39

0.05 0.20 1.56 0.20

0.10 0.39 3.13 0.39

NFLX OFLX

Alcaligenes faecalis (15)

AM-1155 CPFX NFLX OFLX

Branhamella catarrhalis (20)

AM-llSS

CPFX NFLX OFLX

Haemophilus influenzae (16)

AM-llSS

CPFX NFLX OFLX

0.78-25 0.78-100 3.13->100 0.78-50 0.025-0.05 0.025-0.05 0.10-0.20 0.05-0.10 0.0125-0.025 0.0125-0.025 0.05-0.10 0.025-0.05

25 25 50 100

6.25 25 100 25 0.025 0.025 0.20 0.10

100 50 >100 >100

12.5 25 >100 25 0.05 0.05 0.20 0.10

0.025 0.0125 0.025 0.025 0.10 0.05 0.05 0.05 Continued on following page

2112

HOSAKA ET AL.

ANTIMICROB. AGENTS CHEMOTHER.

TABLE 1-Continued MIC (1g/ml)

Organism

(no. of isolates)

Drug

Range

50%

90%

Neisseria gonorrhoeae (10)

AM-1155 CPFX NFLX OFLX

0.0063-0.0125 0.0063-0.0125 0.025-0.05 0.0125-0.025

0.0063 0.0063 0.05 0.0125

0.0125 0.0063 0.05 0.025

Flavobacterium spp. (26)

AM-1155 CPFX NFLX OFLX

0.20-> 100 0.78-> 100 1.56-> 100 0.78-> 100

Campylobacterjejuni (21)

AM-1155 CPFX NFLX OFLX

0.05-0.20 0.05-0.39 0.20-1.56 0.10-0.78

Bacteroides fragilis (23)

AM-1155 CPFX NFLX OFLX

0.39-3.13 3.13-> 100 25-> 100 1.56-25

a

CPFX, ciprofloxacin;

>100 > 100 > 100 > 100

0.10 0.20 0.39 0.39

0.20 0.39 0.78 0.39

0.39 12.5 100 3.13

1.56 25 >100 12.5

NFLX, norfloxacin; OFLX, ofloxacin.

natants were obtained. Urine was collected for 24 h after administration of drug. Samples were assayed for bioactivity by the cup diffusion procedure with Bacillus subtilis ATCC 6633 or E. coli NIHJ JC-2 as the test organism by the method described by Oomori et al. (14).

RESULTS Antibacterial activit. The antibacterial activity of AM1155 was determined for 516 clinical isolates and was comTABLE 2. Antibacterial activities of AM-1155 against the obligately anaerobic bacteria L. pneumophila and M. pneumoniae Organism

AM-1155

CPFX

NFLX

0.39 0.39 0.39 1.56 1.56 0.78 0.78 3.13 3.13 0.78 0.39 0.39 0.20 3.13 0.78 3.13 1.56

6.25 3.13 6.25 6.25 25 12.5 1.56 12.5 12.5 1.56 0.78 0.78 0.39 12.5 0.78 12.5 1.56

50 25 50 50 >100 >100 25 100 100 6.25 6.25 6.25 3.13 100 1.56 >100 25

0.39

0.78

3.13

0.39 100 100

CPFX, ciprofloxacin; NFLX, norfloxacin.

pared with those of ciprofloxacin, norfloxacin, and ofloxacin (Tables 1 and 2). Table 1 summarizes the MIC ranges and the MICs for 50 and 90% of the strains tested (MIC50s and MIC90s, respectively). AM-1155 was very active against S. aureus including methicillin-resistant strains, Staphylococcus epidermidis, streptococci, E. coli, K pneumoniae, Enterobacter cloacae, Proteus spp., Morganella morganii, Acinetobacter calcoaceticus, Branhamella catarrhalis, H. influenzae, Neisseria gonorrhoeae, and C. jejuni, with MIC90s of 800 100 800 NA, nali-

ANTIBACTERIAL ACTIVITY OF AM-1155

VOL. 36, 1992

P. aeruginosa IF0 1 2689

E. coli NIHJ JC-2

S. aureus 209P 10 E 9 0 8

10 9

10 9

8

7

7

8 7

6 U 5 U

6 5

6

039 Ig/mi

5

0.78I g/ml

4 3 2 1

4

CR cn a

4 na 3 2 :3 S

._

1 0

0.20 Lg/ml

g/ml

\01

limit of assay L

-2 0 2 4

6

Time (h)

24

0l

Lg/m

2 6.25

IX0.025 g/m

limit of _I L ' | *

*

I

2

4

6

-2 0

Control

3

\\ 0.0125 g/ml (MIC) 0.05

2113

1

assay .

24

_

313

-

_I,L

0

t

\.t56 ILg/ml (MJIC)

limit of assay I

I

24

4 6

-202

Time (h)

Time (h)

FIG. 2. Bactericidal activity of AM-1155 against S. aureus, E. coli, and P. aeruginosa.

cept P. aeruginosa, and B. fragilis and had slightly greater activity against enterococci, streptococci, and C. jejuni. The activity of AM-1155 against the majority of the members of

the family Enterobacteriaceae, B. catarrhalis, H. influenand N. gonorrhoeae was almost the same as that of ciprofloxacin. AM-1155 was more potent than ofloxacin and norfloxacin against these strains. AM-1155 inhibited 50% of norfloxacin-resistant S. aureus and S. epidermidis strains at concentrations of 3.13 and 1.56 jig/ml, respectively. Against P. aeruginosa, the activity of AM-1155 was similar to those of norfloxacin and ofloxacin, but it was fourfold less than that of ciprofloxacin. AM-1155 was inactive against norfloxacin- and ciprofloxacin-resistant P. aeruginosa. The antibacterial activities of AM-1155 against the obligately anaerobic organisms L. pneumophila and M. pneumoniae are given in Table 2. AM-1155 inhibited Bacteroides spp., Fusobacterium spp., Eubacterium spp., Propionibacterium acnes, Peptococcus magnus, Clostridium spp., Peptostreptococcus spp., and VeilloneUla paruvula at concentrations of 0.20 to 3.13 jig/ml. The MICs of AM-1155 for L. pneumophila GIFU2522 and M. pneumoniae Mac were s0.0063 and 0.10 ,g/ml, respectively. In vitro activity against quinolone-resistant mutants. The drug susceptibilities of DNA gyrase mutants (KEA12 and MH5 of E. coli [8], KH4023 of P. aeruginosa [10]) and outer membrane permeability mutants (KEA13 and KE7 of E. coli [7, 8], KH4013E and KH4014a of P. aeruginosa [2, 10]) are given in Table 3. E. coli KEA12 (norA) and MH5 (gyrA) and P. aeruginosa KH4023 (nfxA) were fourfold less susceptible than the respective parent strains to AM-1155. Their susceptibilities to other quinolones such as ciprofloxacin and norfloxacin were also decreased. E. coli KEA13 and KE7, OmpF-deficient mutants, were two- to fourfold less susceptible than the parent strains to quinolones, including AM1155. The MICs of AM-1155 for P. aeruginosa KH4013E (nfxB) and KH4014a (nfxcC) were 1.56 and 3.13 ,g/ml, respectively, which were four and eight times the MIC, respectively, for the parent strain PA04009. MBCs. The MBCs of AM-1155 and ciprofloxacin for 10 zae,

clinical isolates each of S. aureus, E. coli, and P. aeruginosa were determined. There was no difference or only a twofold difference in the MIC90s and MBCgos. The bactericidal activity of AM-1155 was similar to that of ciprofloxacin (data not shown). Killing-curve studies. Killing-curve studies were performed with AM-1155 against S. aureus 209P, E. coli NIHJ JC-2, and P. aeruginosa IF012689. The rapid bactericidal activity of AM-1155 against these bacteria at concentrations greater than the MIC is shown in Fig. 2. More than 99% of the initial number of viable bacteria were killed within 1 or 2 h of incubation with AM-1155 at concentrations equal to or twice the MIC. Inhibitory effects on DNA gyrase. The inhibitory effects of AM-1155 and ciprofloxacin on DNA gyrase from test strains are given in Table 4. The 50% inhibitory concentrations (IC50s) of AM-1155 on the supercoiling activities of DNA gyrases from M. luteus ATCC 4698, E. coli KL-16, and P. aeruginosa PA01840 were 8.3, 0.38, and 23.3 ,ug/ml, respectively. The DNA gyrases from gyrA mutants of E. coli KL-16 and P. aeruginosa PA01840 were resistant to AMTABLE 4. Inhibition of supercoiling activity of DNA gyrases by AM-1155 DNA gyrase from:

M. luteus ATCC 4698

AM-1155 MIC IC50

CPFXa

MIC

IC50 (,ug/ml) 14.0

(1lg/ml) 0.025

(11g/ml) 8.3

(pg/ml) 0.20

0.025 0.39

0.38 13.5

0.025 0.39

E. coli

KL-16 (wild type) N-51 (gyrA)

0.27 23.9

P. aeruginosa

PA01840 (wild type)

PKH-T477 (gyrA) a CPFX, ciprofloxacin.

1.56 12.5

23.3 187

0.39 6.25

8.0 >400

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HOSAKA ET AL.

ANTIMICROB. AGENTs CHEMOTHER.

TABLE S. Frequencies of spontaneous mutants resistant to AM-1155, ciprofloxacin, and norfloxacin Organism

Original (ILg/min)MIC

AM-1155

0.05

CPFX NFLX

0.20 0.78

Mutation frequency

In vitro and in vivo antibacterial activities of AM-1155, a new 6-fluoro-8-methoxy quinolone.

AM-1155 is a new quinolone with a wide spectrum of antibacterial activity against various bacteria including anaerobes and Mycoplasma pneumoniae. AM-1...
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