ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUlY 1992, p. 1491-1498 0066-4804/92/071491-08$02.00/0 Copyright © 1992, American Society for Microbiology
Vol. 36, No. 7
Antimicrobial Activity of DU-6859, a New Potent Fluoroquinolone, against Clinical Isolates KENICHI SATO,* KAZUKI HOSHINO, MAYUMI TANAKA, ISAO HAYAKAWA, AND YASUAKI OSADA Exploratory Research Laboratories I, Daiichi Pharnaceutical Co., Ltd., Edogawa-ku, Tokyo, 134, Japan Received 11 December 1991/Accepted 25 April 1992
DU-6859, (-)-7-[(7S)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6-fluoro-1- [(LR,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinolone-3-carboxylic acid, is a new fluoroquinolone with antibacterial activity which is significantly better than those of currently available quinolones. The MICs for 90%o of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis clinical isolates (MIC90s) were 0.1, 3.13, 0.1, and 0.39 ,ug/ml, respectively. MICsos of DU-6859 against quinolone-resistant, methicillin-resistant S. aureus were 8-, 32-, 64-, and 128-fold lower than those of tosufloxacin and sparfloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DU-6859 inhibited the growth of all strains of Streptococcus pneumoniae and Streptococcus pyogenes at 0.1 and 0.2 ,ug/ml, respectively, and was more active against enterococci than the other quinolones tested. Although the activity of DU-6859 against Pseudomonas aeruginosa was roughly comparable to that of ciprofloxacin at the MIC,o level, it was fourfold more active than ciprofloxacin at the MIC90 level. DU-6859 was also more active against other glucose-nonfermenting bacteria, Haemophilus influenzae, Moraxela catarrhalis, and Neisseria gonorrhoeae, than the other drugs tested. Strains of Bacteroidesfragilis and Peptostreptococcus spp. were susceptible to DU-6859; MIC90s were 0.39 and 0.2 ,ug/ml, respectively. DU-6859 generally showed activities twofold or greater than those of ciprofloxacin and the other drugs against almost all members of the family Enterobacteriaceae. The action of DU-6859 against the clinical isolates was bactericidal at concentrations near the MICs. DU-6859 activity was not affected by different media, pH, inoculum size, or human serum but was decreased in human urine. Because of their broad antibacterial spectra, bactericidal activities, and good pharmacokinetics (12, 16, 17), the new fluoroquinolones, such as ofloxacin and ciprofloxacin, are used for the treatment of various infections. Recently, however, quinolone-resistant staphylococcal and pseudomonal strains have increased in frequency (10, 14, 18), and activities of these drugs against Streptococcus pneumoniae, enterococci, and Bacteroides firagilis may be borderline (17, 18), limiting their use in the treatment of bacteremia, sepsis, and respiratory tract infections. DU-6859 is a new fluoroquinolone with the chemical structure (-)-7-[(75)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6fluoro-1-[(1R,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinolone-3-carboxylic acid synthesized by Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan) (Fig. 1). In this study, we compared the in vitro activities of DU-6859 with those of ciprofloxacin, ofloxacin, sparfloxacin, tosufloxacin, fleroxacin, and lomefloxacin against freshly isolated bacteria, adding new data to a previous study (11). We also studied the bactericidal activity and the effects of different media, inoculum size, pH, human serum, and urine on the activity of DU-6859.
(Meiji Seika Co., Ltd., Tokyo, Japan), and clindamycin phosphate (Sigma) were obtained commercially. Organisms. A total of 1,393 strains, which were isolated from patients at six medical centers located in the Tokyo and Tohoku areas in Japan from 1990 to 1991, were randomly used for this study (one isolate per patient). Two reference strains (Escherichia coli NIH JC-2 and Staphylococcus aureus FDA209P) were included as internal controls in all parts of the study. Determination of MICs. The MICs were determined by standard agar dilution methods (6) with Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.). Mueller-Hinton agar supplemented with 5% sheep blood and 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) was used for Haemophilus influenzae, streptococci, and Moraxella catarrhalis. GC agar (Difco) supplemented with 1% hemoglobin and 1% IsoVitaleX was used for Neisseria gonorrhoeae, while GAM agar (Nissui Seiyaku Co., Ltd., Tokyo, Japan) was used for anaerobic bacteria. One loopful (5 ,ul) of an inoculum corresponding to about 5 x 103 CFU per spot was inoculated onto drug-containing agar plates, and the plates were incubated for 18 h at 37°C, except for B. fragilis and Peptostreptococcus spp., which were incubated for 24 h. H. influenzae and N. gonorrhoeae were incubated under conditions of 10% CO2. B. fragilis and Peptostreptococcus spp. were incubated in an anaerobic cabinet. The MIC was defined as the lowest drug concentration which prevented visible growth of bacteria. The effects of medium, inoculum size, pH, and human serum on DU-6859 activity were also performed by the Mueller-Hinton agar dilution method. The effect of human urine was determined by the macrodilution tube method (6) using 100% human urine. The final inoculum size was 5 x 105 CFU/ml. Determination of MBCs. Bactericidal tests were performed
MATERIALS AND METHODS
Antibacterial agents. DU-6859 and ofloxacin (OFLX) were synthesized at the Exploratory Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., as were ciprofloxacin (CPFX), sparfloxacin (SPFX), tosufloxacin (TFLX), fleroxacin (FLRX), and lomefloxacin (LFLX). Methicillin sodium (Sigma Chemical Co., St. Louis, Mo.), ampicillin sodium *
Corresponding author. 1491
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ANTIMICROB. AGENTS CHEMOTHER.
SATO ET AL.
F,
CI
FIG. 1. Chemical structure of DU-6859.
by the micro-broth-dilution method described by Taylor et al. (15) with an inoculum of 5 x 105 CFU/ml. The MBC was defined as the lowest concentration of each drug which exhibited .99.9% bactericidal activity by the National Committee for Clinical Laboratory Standards method (6). RESULTS Antibacterial activity. Table 1 compares the activities of DU-6859 against gram-positive and -negative bacteria with those of the reference drugs. The MIC of DU-6859 against 90% of methicillin-susceptible S. aureus (MSSA) and Staphylococcus epidernidis (MSSE) (MIC90) was 0.1 ,ug/ml for both. Against methicillin-resistant (methicillin MIC, 212.5 ,ug/ml) S. aureus (MRSA) and S. epidermidis (MRSE), MIC90s were 0.2 and 0.39 ,ug/ml, respectively. The activity of DU-6859 against MSSA was comparable to that of TFLX and twofold higher than that of SPFX at the MICJ.. DU-6859 was also twofold more active than SPFX and TFLX and eightfold more active than OFLX and CPFX against MSSE. Against quinolone-susceptible MRSA, the activity of DU6859 was also comparable to those of SPFX and TFLX and was at least eightfold more active than the other drugs. Furthermore, DU-6859 showed the highest activity against quinolone-resistant MRSA (OFLX MIC, >6.25 ,ig/ml) among the drugs tested. Against these strains, the activity was 8-, 32-, 64-, and 128-fold higher than those of TFLX and SPFX, OFLX and FLRX, CPFX, and LFLX, respectively, at the M'C50. DU-6859 was also 8- and 16-fold more potent than SPFX and TFLX, respectively, and was at least 64-fold more potent than OFLX, CPFX, FLRX, and LFLX against MRSE. The activities of DU-6859 against streptococci and enterococci were at least fourfold or greater than those of other drugs. MIC90s for S. pneumoniae, Streptococcus pyogenes, and Enterococcus faecalis were 0.1, 0.1, and 0.39 ,ug/ml, respectively. In particular, DU-6859 inhibited all strains, including ampicillin-resistant strains (MIC, .0.39 jig/ml) of pneumococci, at a concentration of 0.1 ,ug/ml. Against Peptostreptococcus spp., DU-6859 activity was similar to that of clindamycin and was 4-fold higher than SPFX and TFLX and 32-fold higher than those of OFLX and CPFX. Against various members of the family Enterobacteriaceae (excepting Klebsiella oxytoca and Proteus mirabilis), DU-6859 activity was twofold or greater than those of CPFX and TFLX at the MIC90 (Table 1). Against K oxytoca and P. mirabilis, the activity of DU-6859 was almost equal to those of CPFX and TFLX. DU-6859 inhibited most (90%)
isolates of E. coli, KIlebsiella pneumoniae, K oxytoca, P. mirabilis, and Morganella morganii at 0.05, 0.20, 0.05, 0.10, and 0.20 ,ug/ml, respectively. At the MIC90, DU-6859 activity was roughly comparable to that of CPFX and 2-fold higher than that of TFLX against quinolone-susceptible Pseudomonas aeruginosa; however, against quinolone-resistant strains, DU-6859 was 4-fold more active than CPFX and 16-fold or greater more active than the other drugs. Other glucose-nonfermenting gramnegative bacteria, including Xanthomonas maltophilia and Acinetobacter calcoaceticus and excepting Alcaligenes faecalis, were mostly susceptible to DU-6859, with MIC90s of 0.78 and 0.39 ,ug/ml, respectively. DU-6859 activity against A. faecalis was less than those against other glucosenonfermenting bacteria. H. influenzae, M. catarrhalis, and N. gonorrhoeae were susceptible to DU-6859, the MIC90s for these strains being 0.006, 0.025, and 0.025 ,ug/ml, respectively. DU-6859 was equally as active as SPFX and 2- to 16-fold more active than TFLX, OFLX, CPFX, FLRX, and LFLX against M. catarrhalis. Against H. influenzae, DU-6859 activity was 2-, 8-, and 16-fold higher than those of SPFX and TFLX, CPFX, and OFLX, FLRX and LFLX, respectively. Against N. gonorrhoeae, DU-6859 was 4-, 8-, 16-, and 32-fold more active than SPFX and TFLX, CPFX, OFLX, and FLRX and LFLX, respectively. Strains of B. fragilis were highly susceptible to DU-6859; the MIC50 and MIC. were 0.10 and 0.39 ,ug/ml, respectively. At the MIC90, DU-6859 was at least 4-, 32-, and 64-fold more active than SPFX and TFLX, OFLX, and other drugs (including clindamycin), respectively. The interpretive MICs of DU-6859 for E. coli NIH JC-2 and S. aureus FDA209P were 0.006 and 0.025 jig/ml, respectively. Factors affecting activity. The activities of DU-6859 against S. aureus FDA 209P, E. coli NIH JC2, and P. aeruginosa PAO1 were nearly the same in any of five different media; namely Mueller-Hinton, nutrient, heart infusion, brain heart infusion, and tryptose blood agar (data not shown). Varying the pH of Mueller-Hinton agar between 6 and 8, adding 10, 25, and 50% human serum to the medium, or increasing the inoculum size from 103 to 107 CFU had no significant effect on the activity of DU-6859 against each strain (data not shown). However, its activity in the Mueller-Hinton agar at pH5 and in human urine decreased to two- to eightfold, as did those of the other drugs tested (data not shown). Bactericidal activity. MICs and MBCs of DU-6859 against 10 or 20 strains of E. coli, quinolone-susceptible P. aeruginosa (OFLX MIC, 6.25 ,ug/ml), MSSA, and MRSA are shown in Table 2. MBC50s and MBC90s of DU-6859 against each species were similar to the MICs, indicating that this compound is highly bactericidal. DISCUSSION DU-6859 is one of the recently developed quinolones (others include PD127,391 and WIN57273)(4, 7, 8) which have more potent in vitro activities against staphylococci, streptococci, and enterococci than related drugs, including SPFX and TFLX. They possess more potent activities than OFLX and CPFX against gram-positive bacteria (1, 5). Moreover, DU-6859 shows excellent activity against members of the family Enterobacteriaceae, P. aeruginosa, M. catarrhalis, H. influenzae, N. gonorrhoeae, and B. fragilis, known clinically as significant pathogens. The difference in
ANTIMICROBIAL ACTIVITY OF DU-6859, A NEW QUINOLONE
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TABLE 1. Antimicrobial activities of DU-6859 and other related quinolones against clinical isolates Organism (no. of isolates)
MIC (,ug/ml)
Drug
Range
Staphylococcus aureus Methicillin susceptibiea (93)
50%o
90%o
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Methicillin
0.013-0.78 0.2-25 0.2-50 0.025-12.5 0.025-12.5 0.39->100 0.39-100 1.56-6.25
0.05 0.39 0.78 0.05 0.1 0.78 0.78 3.13
0.1 0.78 0.78 0.1 0.2 1.56 0.78 3.13
Methicillin resistant,' quinolone susceptibleC (49)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Methicillin
0.013-0.2 0.2-3.13 0.2-12.5 0.025-1.56 0.013-0.78 0.39-12.5 0.39-12.5 12.5->100
0.05 0.39 0.78 0.05 0.05 0.78 0.78 >100
0.2 1.56 6.25 0.39 0.2 6.25 6.25 >100
Methicillin resistant,' quinolone-resistant' (52)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.2-3.13 6.25-100 3.13-> 100 0.78-> 100 0.78-12.5 6.25-> 100 12.5->100 50-> 100
0.78 25 50 6.25 6.25 100 25 >100
3.13 100 >100 >100 12.5 >100 >100 >100
0.025-0.2 0.39-6.25 0.2-6.25 0.05-3.13 0.1-3.13 0.78-50 0.39-25 0.78-6.25
0.05 0.39 0.39 0.1 0.2 0.78 0.78 3.13
0.1 0.78 0.78 0.2 0.2 1.56 1.56 6.25
0.013-0.78 0.39-50 0.2-50 0.05-12.5 0.1-12.5 0.78-100 0.39->100 12.5->100
0.05 0.78 0.39 0.1 0.2 0.78 0.78 50
0.39 25 25 6.25 3.13 100 >100 >100
Methicillin
Staphylococcus epidermidis Methicillin susceptiblea (34)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Methicillin Methicillin resistant" (41)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Methicillin Streptococcus pneumoniae (49)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Ampicillin
Streptococcus,pyogenes (25)
DU-6859 OFLX CPFX
TFLX SPFX LFLX FLRX
Ampicillin
0.025-0.1 0.78-3.13 0.39-3.13 0.1-0.78 0.1-0.78 1.56-12.5 3.13-12.5 100 0.39-> 100 0.2-25 0.39-50 1.56-> 100 1.56-> 100
0.39 6.25 3.13 1.56 0.78 12.5 12.5
1.56 100 100 25 50 >100 >100
Peptostreptococcus spp. (24)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Clindamycin
0.1-0.39 1.56-6.25 1.56-6.25 0.39-0.78 0.78-1.56 6.25-25 6.25-25 0.05-0.78
0.1 3.13 3.13 0.39 0.78 12.5 12.5 0.2
0.2 6.25 6.25 0.78 0.78 25 25 0.2
Escherichia coli (107)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
.0.003-1.56 .0.003-50 .0.003-25 .0.003-25
Enterobacter cloacae (50)
Klebsiella pneumoniae (50)
0.006-25 s0.003-> 100 .0.003-> 100
0.013 0.1 0.025 0.025 0.025 0.1 0.1
0.05 0.39 0.1 0.1 0.1 0.39 0.39
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.006-6.25 0.05-100 0.006-100 .0.003-> 100 0.013-100 0.05-> 100 0.05-> 100
0.025 0.2 0.05 0.05 0.05 0.2 0.2
1.56 25 12.5 12.5 12.5 50 25
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.013-1.56 0.1-25 0.025-6.25
.0.003-3.13 0.013-6.25 0.1-25 0.1-25
0.05 0.1 0.05 0.05 0.05 0.2 0.2
0.2 3.13 0.39 0.39 0.78 1.56 1.56
Klebsiella oxytoca (50)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.006-0.39 0.1-1.56 0.013-0.78 0.013-0.78 0.013-1.56 0.1-3.13 0.1-3.13
0.025 0.2 0.05 0.025 0.05 0.2 0.2
0.05 0.2 0.05 0.05 0.1 0.2 0.2
Serratia marcescens (50)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.39 0.78 0.39 0.78 1.56 0.78 0.78
3.13 50 12.5 25 25 50 25
0.05-6.25 0.2-50 0.05-25 0.1-> 100 0.2-50 0.2-100 0.2-50
Continued on following page
ANTIMICROBIAL ACTIVITY OF DU-6859, A NEW QUINOLONE
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TABLE 1-Continued Organism (no. of isolates)
Citrobacterffreundii (50)
Drug
Range
MIC (,ug/ml) 50%
90%
0.025-12.5 0.1->100 0.013->100 0.025->100 0.025->100 0.2->100 0.2-> 100
0.39 0.78 0.2 0.39 1.56 1.56 0.78
6.25 >100 >100 >100 >100 >100 >100
0.025-0.1 0.1-0.78 0.025-0.2 0.1-0.78 0.05-0.78 0.2-0.78 0.2-0.39
0.05 0.39 0.1 0.2 0.39 0.39 0.2
0.1 0.39 0.1 0.39 0.39 0.78 0.39
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.013-12.5 0.1-100
0.025->100 0.05->100 0.05-> 100 0.2->100 0.1-> 100
0.05 0.2 0.05 0.2 0.39 0.2 0.2
12.5 100 >100 >100 >100 >100 100
Providencia rettgeri (25)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.025-6.25 0.1->100 0.025->100 0.1->100 0.1->100 0.2->100 0.1->100
1.56 12.5 25 6.25 6.25 25 12.5
6.25 >100 >100 25 >100 >100 >100
Providencia stuartii (25)
DU-6859 OFLX CPFX TFLX SPFX
0.1-6.25 0.78-100 0.1->100 0.1-25 0.2-25 0.39->100 0.2-50
0.2 1.56 0.78 0.39 0.39 1.56 0.78
3.13 25 50 12.5 6.25 50 50
0.013-6.25 0.1-100 0.013-50 0.05-6.25 0.1-50 0.1->100 0.1->100
0.025 0.2 0.05 0.2 0.2 0.2 0.1
0.2 1.56 0.78 1.56 1.56 3.13 1.56
0.05-0.78 0.39-3.13 0.05-0.78 0.1-0.78 0.2-3.13 0.39-6.25 0.2-6.25
0.2 1.56 0.39 0.39 0.78 1.56 1.56
0.39 3.13 0.39 0.78 1.56 3.13 3.13
0.39-50 6.25-> 100 0.39-> 100
3.13 25 3.13 6.25 12.5 50 25
DU-6859 OFLX CPFX TFLX
SPFX LFLX FLRX
Proteus mirabilis (50)
DU-6859
OFLX CPFX TFLX SPFX LFLX FLRX
Proteus vulgaris (50)
LFLX FLRX
Morganella morganii (50)
DU-6859 OFLX CPFX
TFLX SPFX LFLX FLRX
Pseudomonas aeruginosa
Quinolone susceptiblec (57)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Quinolone resistantd (46)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.78->100 1.56->100 3.13->100
3.13-> 100
12.5 >100 50 >100 >100 >100 >100
Continued on following page
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1-Continued
Organism (no. of isolates)
Drug
MIC (jig/ml)
Range
50%
90%
Pseudomonas cepacia (25)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.1-3.13 0.78-25 0.78-25 0.39-25 0.2-25 1.56-50 0.78-25
1.56 12.5 3.13 6.25 3.13 12.5 6.25
3.13 25 12.5 25 25 50 25
Xanthomonas maltophilia (24)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.05-0.78 0.78-12.5 0.78-12.5 0.2-3.13 0.1-3.13 0.78-12.5 0.78-6.25
0.39 3.13 3.13 0.39 0.78 3.13 1.56
0.78 6.25 6.25 1.56 1.56 6.25 3.13
Haemophilus influenzae (44)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
.0.003 0.05 0.013 0.006 .0.003 0.1 0.05
0.006 0.1 0.05 0.013 0.013 0.1 0.1
0.025 0.39 0.2 0.1 0.1 0.78 0.78 1.56
Neisseria gonorrhoeae (25)
100 >100
25 >100 >100 >100 >100 >100 >100
Continued on following page
ANTIMICROBIAL ACTIVITY OF DU-6859, A NEW QUINOLONE
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TABLE 1-Continued MIC (,ug/ml)
Drug
Organism (no. of isolates)
Range
Alcaligenes denitrificans subsp. xylosoxydans (25)
Bacteroides fiagillis (23)
50%
90%
3.13
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.2-6.25
0.78
1.56-> 100 1.56-> 100 0.39-> 100 0.39-50 1.56->100 1.56-100
25 12.5 12.5 6.25 25 6.25
100 100 > 100 50 100 50
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Clindamycin
0.05-0.78 1.56-50 3.13-100 0.39-12.5 0.78-25 6.25-50 6.25-100 0.05->100
0.1 3.13 6.25 0.78 1.56 12.5 12.5 0.2
12.5 50 1.56 1.56 25 25 > 100
0.39
a Methicillin susceptible, methicillin MIC of
Vol. 36, No. 7
Antimicrobial Activity of DU-6859, a New Potent Fluoroquinolone, against Clinical Isolates KENICHI SATO,* KAZUKI HOSHINO, MAYUMI TANAKA, ISAO HAYAKAWA, AND YASUAKI OSADA Exploratory Research Laboratories I, Daiichi Pharnaceutical Co., Ltd., Edogawa-ku, Tokyo, 134, Japan Received 11 December 1991/Accepted 25 April 1992
DU-6859, (-)-7-[(7S)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6-fluoro-1- [(LR,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinolone-3-carboxylic acid, is a new fluoroquinolone with antibacterial activity which is significantly better than those of currently available quinolones. The MICs for 90%o of methicillin-susceptible and -resistant Staphylococcus aureus and Staphylococcus epidermidis clinical isolates (MIC90s) were 0.1, 3.13, 0.1, and 0.39 ,ug/ml, respectively. MICsos of DU-6859 against quinolone-resistant, methicillin-resistant S. aureus were 8-, 32-, 64-, and 128-fold lower than those of tosufloxacin and sparfloxacin, ofloxacin and fleroxacin, ciprofloxacin, and lomefloxacin, respectively. DU-6859 inhibited the growth of all strains of Streptococcus pneumoniae and Streptococcus pyogenes at 0.1 and 0.2 ,ug/ml, respectively, and was more active against enterococci than the other quinolones tested. Although the activity of DU-6859 against Pseudomonas aeruginosa was roughly comparable to that of ciprofloxacin at the MIC,o level, it was fourfold more active than ciprofloxacin at the MIC90 level. DU-6859 was also more active against other glucose-nonfermenting bacteria, Haemophilus influenzae, Moraxela catarrhalis, and Neisseria gonorrhoeae, than the other drugs tested. Strains of Bacteroidesfragilis and Peptostreptococcus spp. were susceptible to DU-6859; MIC90s were 0.39 and 0.2 ,ug/ml, respectively. DU-6859 generally showed activities twofold or greater than those of ciprofloxacin and the other drugs against almost all members of the family Enterobacteriaceae. The action of DU-6859 against the clinical isolates was bactericidal at concentrations near the MICs. DU-6859 activity was not affected by different media, pH, inoculum size, or human serum but was decreased in human urine. Because of their broad antibacterial spectra, bactericidal activities, and good pharmacokinetics (12, 16, 17), the new fluoroquinolones, such as ofloxacin and ciprofloxacin, are used for the treatment of various infections. Recently, however, quinolone-resistant staphylococcal and pseudomonal strains have increased in frequency (10, 14, 18), and activities of these drugs against Streptococcus pneumoniae, enterococci, and Bacteroides firagilis may be borderline (17, 18), limiting their use in the treatment of bacteremia, sepsis, and respiratory tract infections. DU-6859 is a new fluoroquinolone with the chemical structure (-)-7-[(75)-amino-5-azaspiro(2,4)heptan-5-yl]-8-chloro-6fluoro-1-[(1R,2R)-cis-2-fluoro-1-cyclopropyl]-1,4-dihydro-4-oxoquinolone-3-carboxylic acid synthesized by Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan) (Fig. 1). In this study, we compared the in vitro activities of DU-6859 with those of ciprofloxacin, ofloxacin, sparfloxacin, tosufloxacin, fleroxacin, and lomefloxacin against freshly isolated bacteria, adding new data to a previous study (11). We also studied the bactericidal activity and the effects of different media, inoculum size, pH, human serum, and urine on the activity of DU-6859.
(Meiji Seika Co., Ltd., Tokyo, Japan), and clindamycin phosphate (Sigma) were obtained commercially. Organisms. A total of 1,393 strains, which were isolated from patients at six medical centers located in the Tokyo and Tohoku areas in Japan from 1990 to 1991, were randomly used for this study (one isolate per patient). Two reference strains (Escherichia coli NIH JC-2 and Staphylococcus aureus FDA209P) were included as internal controls in all parts of the study. Determination of MICs. The MICs were determined by standard agar dilution methods (6) with Mueller-Hinton agar (Difco Laboratories, Detroit, Mich.). Mueller-Hinton agar supplemented with 5% sheep blood and 1% IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.) was used for Haemophilus influenzae, streptococci, and Moraxella catarrhalis. GC agar (Difco) supplemented with 1% hemoglobin and 1% IsoVitaleX was used for Neisseria gonorrhoeae, while GAM agar (Nissui Seiyaku Co., Ltd., Tokyo, Japan) was used for anaerobic bacteria. One loopful (5 ,ul) of an inoculum corresponding to about 5 x 103 CFU per spot was inoculated onto drug-containing agar plates, and the plates were incubated for 18 h at 37°C, except for B. fragilis and Peptostreptococcus spp., which were incubated for 24 h. H. influenzae and N. gonorrhoeae were incubated under conditions of 10% CO2. B. fragilis and Peptostreptococcus spp. were incubated in an anaerobic cabinet. The MIC was defined as the lowest drug concentration which prevented visible growth of bacteria. The effects of medium, inoculum size, pH, and human serum on DU-6859 activity were also performed by the Mueller-Hinton agar dilution method. The effect of human urine was determined by the macrodilution tube method (6) using 100% human urine. The final inoculum size was 5 x 105 CFU/ml. Determination of MBCs. Bactericidal tests were performed
MATERIALS AND METHODS
Antibacterial agents. DU-6859 and ofloxacin (OFLX) were synthesized at the Exploratory Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., as were ciprofloxacin (CPFX), sparfloxacin (SPFX), tosufloxacin (TFLX), fleroxacin (FLRX), and lomefloxacin (LFLX). Methicillin sodium (Sigma Chemical Co., St. Louis, Mo.), ampicillin sodium *
Corresponding author. 1491
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ANTIMICROB. AGENTS CHEMOTHER.
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F,
CI
FIG. 1. Chemical structure of DU-6859.
by the micro-broth-dilution method described by Taylor et al. (15) with an inoculum of 5 x 105 CFU/ml. The MBC was defined as the lowest concentration of each drug which exhibited .99.9% bactericidal activity by the National Committee for Clinical Laboratory Standards method (6). RESULTS Antibacterial activity. Table 1 compares the activities of DU-6859 against gram-positive and -negative bacteria with those of the reference drugs. The MIC of DU-6859 against 90% of methicillin-susceptible S. aureus (MSSA) and Staphylococcus epidernidis (MSSE) (MIC90) was 0.1 ,ug/ml for both. Against methicillin-resistant (methicillin MIC, 212.5 ,ug/ml) S. aureus (MRSA) and S. epidermidis (MRSE), MIC90s were 0.2 and 0.39 ,ug/ml, respectively. The activity of DU-6859 against MSSA was comparable to that of TFLX and twofold higher than that of SPFX at the MICJ.. DU-6859 was also twofold more active than SPFX and TFLX and eightfold more active than OFLX and CPFX against MSSE. Against quinolone-susceptible MRSA, the activity of DU6859 was also comparable to those of SPFX and TFLX and was at least eightfold more active than the other drugs. Furthermore, DU-6859 showed the highest activity against quinolone-resistant MRSA (OFLX MIC, >6.25 ,ig/ml) among the drugs tested. Against these strains, the activity was 8-, 32-, 64-, and 128-fold higher than those of TFLX and SPFX, OFLX and FLRX, CPFX, and LFLX, respectively, at the M'C50. DU-6859 was also 8- and 16-fold more potent than SPFX and TFLX, respectively, and was at least 64-fold more potent than OFLX, CPFX, FLRX, and LFLX against MRSE. The activities of DU-6859 against streptococci and enterococci were at least fourfold or greater than those of other drugs. MIC90s for S. pneumoniae, Streptococcus pyogenes, and Enterococcus faecalis were 0.1, 0.1, and 0.39 ,ug/ml, respectively. In particular, DU-6859 inhibited all strains, including ampicillin-resistant strains (MIC, .0.39 jig/ml) of pneumococci, at a concentration of 0.1 ,ug/ml. Against Peptostreptococcus spp., DU-6859 activity was similar to that of clindamycin and was 4-fold higher than SPFX and TFLX and 32-fold higher than those of OFLX and CPFX. Against various members of the family Enterobacteriaceae (excepting Klebsiella oxytoca and Proteus mirabilis), DU-6859 activity was twofold or greater than those of CPFX and TFLX at the MIC90 (Table 1). Against K oxytoca and P. mirabilis, the activity of DU-6859 was almost equal to those of CPFX and TFLX. DU-6859 inhibited most (90%)
isolates of E. coli, KIlebsiella pneumoniae, K oxytoca, P. mirabilis, and Morganella morganii at 0.05, 0.20, 0.05, 0.10, and 0.20 ,ug/ml, respectively. At the MIC90, DU-6859 activity was roughly comparable to that of CPFX and 2-fold higher than that of TFLX against quinolone-susceptible Pseudomonas aeruginosa; however, against quinolone-resistant strains, DU-6859 was 4-fold more active than CPFX and 16-fold or greater more active than the other drugs. Other glucose-nonfermenting gramnegative bacteria, including Xanthomonas maltophilia and Acinetobacter calcoaceticus and excepting Alcaligenes faecalis, were mostly susceptible to DU-6859, with MIC90s of 0.78 and 0.39 ,ug/ml, respectively. DU-6859 activity against A. faecalis was less than those against other glucosenonfermenting bacteria. H. influenzae, M. catarrhalis, and N. gonorrhoeae were susceptible to DU-6859, the MIC90s for these strains being 0.006, 0.025, and 0.025 ,ug/ml, respectively. DU-6859 was equally as active as SPFX and 2- to 16-fold more active than TFLX, OFLX, CPFX, FLRX, and LFLX against M. catarrhalis. Against H. influenzae, DU-6859 activity was 2-, 8-, and 16-fold higher than those of SPFX and TFLX, CPFX, and OFLX, FLRX and LFLX, respectively. Against N. gonorrhoeae, DU-6859 was 4-, 8-, 16-, and 32-fold more active than SPFX and TFLX, CPFX, OFLX, and FLRX and LFLX, respectively. Strains of B. fragilis were highly susceptible to DU-6859; the MIC50 and MIC. were 0.10 and 0.39 ,ug/ml, respectively. At the MIC90, DU-6859 was at least 4-, 32-, and 64-fold more active than SPFX and TFLX, OFLX, and other drugs (including clindamycin), respectively. The interpretive MICs of DU-6859 for E. coli NIH JC-2 and S. aureus FDA209P were 0.006 and 0.025 jig/ml, respectively. Factors affecting activity. The activities of DU-6859 against S. aureus FDA 209P, E. coli NIH JC2, and P. aeruginosa PAO1 were nearly the same in any of five different media; namely Mueller-Hinton, nutrient, heart infusion, brain heart infusion, and tryptose blood agar (data not shown). Varying the pH of Mueller-Hinton agar between 6 and 8, adding 10, 25, and 50% human serum to the medium, or increasing the inoculum size from 103 to 107 CFU had no significant effect on the activity of DU-6859 against each strain (data not shown). However, its activity in the Mueller-Hinton agar at pH5 and in human urine decreased to two- to eightfold, as did those of the other drugs tested (data not shown). Bactericidal activity. MICs and MBCs of DU-6859 against 10 or 20 strains of E. coli, quinolone-susceptible P. aeruginosa (OFLX MIC, 6.25 ,ug/ml), MSSA, and MRSA are shown in Table 2. MBC50s and MBC90s of DU-6859 against each species were similar to the MICs, indicating that this compound is highly bactericidal. DISCUSSION DU-6859 is one of the recently developed quinolones (others include PD127,391 and WIN57273)(4, 7, 8) which have more potent in vitro activities against staphylococci, streptococci, and enterococci than related drugs, including SPFX and TFLX. They possess more potent activities than OFLX and CPFX against gram-positive bacteria (1, 5). Moreover, DU-6859 shows excellent activity against members of the family Enterobacteriaceae, P. aeruginosa, M. catarrhalis, H. influenzae, N. gonorrhoeae, and B. fragilis, known clinically as significant pathogens. The difference in
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TABLE 1. Antimicrobial activities of DU-6859 and other related quinolones against clinical isolates Organism (no. of isolates)
MIC (,ug/ml)
Drug
Range
Staphylococcus aureus Methicillin susceptibiea (93)
50%o
90%o
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Methicillin
0.013-0.78 0.2-25 0.2-50 0.025-12.5 0.025-12.5 0.39->100 0.39-100 1.56-6.25
0.05 0.39 0.78 0.05 0.1 0.78 0.78 3.13
0.1 0.78 0.78 0.1 0.2 1.56 0.78 3.13
Methicillin resistant,' quinolone susceptibleC (49)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Methicillin
0.013-0.2 0.2-3.13 0.2-12.5 0.025-1.56 0.013-0.78 0.39-12.5 0.39-12.5 12.5->100
0.05 0.39 0.78 0.05 0.05 0.78 0.78 >100
0.2 1.56 6.25 0.39 0.2 6.25 6.25 >100
Methicillin resistant,' quinolone-resistant' (52)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.2-3.13 6.25-100 3.13-> 100 0.78-> 100 0.78-12.5 6.25-> 100 12.5->100 50-> 100
0.78 25 50 6.25 6.25 100 25 >100
3.13 100 >100 >100 12.5 >100 >100 >100
0.025-0.2 0.39-6.25 0.2-6.25 0.05-3.13 0.1-3.13 0.78-50 0.39-25 0.78-6.25
0.05 0.39 0.39 0.1 0.2 0.78 0.78 3.13
0.1 0.78 0.78 0.2 0.2 1.56 1.56 6.25
0.013-0.78 0.39-50 0.2-50 0.05-12.5 0.1-12.5 0.78-100 0.39->100 12.5->100
0.05 0.78 0.39 0.1 0.2 0.78 0.78 50
0.39 25 25 6.25 3.13 100 >100 >100
Methicillin
Staphylococcus epidermidis Methicillin susceptiblea (34)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Methicillin Methicillin resistant" (41)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Methicillin Streptococcus pneumoniae (49)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Ampicillin
Streptococcus,pyogenes (25)
DU-6859 OFLX CPFX
TFLX SPFX LFLX FLRX
Ampicillin
0.025-0.1 0.78-3.13 0.39-3.13 0.1-0.78 0.1-0.78 1.56-12.5 3.13-12.5 100 0.39-> 100 0.2-25 0.39-50 1.56-> 100 1.56-> 100
0.39 6.25 3.13 1.56 0.78 12.5 12.5
1.56 100 100 25 50 >100 >100
Peptostreptococcus spp. (24)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Clindamycin
0.1-0.39 1.56-6.25 1.56-6.25 0.39-0.78 0.78-1.56 6.25-25 6.25-25 0.05-0.78
0.1 3.13 3.13 0.39 0.78 12.5 12.5 0.2
0.2 6.25 6.25 0.78 0.78 25 25 0.2
Escherichia coli (107)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
.0.003-1.56 .0.003-50 .0.003-25 .0.003-25
Enterobacter cloacae (50)
Klebsiella pneumoniae (50)
0.006-25 s0.003-> 100 .0.003-> 100
0.013 0.1 0.025 0.025 0.025 0.1 0.1
0.05 0.39 0.1 0.1 0.1 0.39 0.39
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.006-6.25 0.05-100 0.006-100 .0.003-> 100 0.013-100 0.05-> 100 0.05-> 100
0.025 0.2 0.05 0.05 0.05 0.2 0.2
1.56 25 12.5 12.5 12.5 50 25
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.013-1.56 0.1-25 0.025-6.25
.0.003-3.13 0.013-6.25 0.1-25 0.1-25
0.05 0.1 0.05 0.05 0.05 0.2 0.2
0.2 3.13 0.39 0.39 0.78 1.56 1.56
Klebsiella oxytoca (50)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.006-0.39 0.1-1.56 0.013-0.78 0.013-0.78 0.013-1.56 0.1-3.13 0.1-3.13
0.025 0.2 0.05 0.025 0.05 0.2 0.2
0.05 0.2 0.05 0.05 0.1 0.2 0.2
Serratia marcescens (50)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.39 0.78 0.39 0.78 1.56 0.78 0.78
3.13 50 12.5 25 25 50 25
0.05-6.25 0.2-50 0.05-25 0.1-> 100 0.2-50 0.2-100 0.2-50
Continued on following page
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TABLE 1-Continued Organism (no. of isolates)
Citrobacterffreundii (50)
Drug
Range
MIC (,ug/ml) 50%
90%
0.025-12.5 0.1->100 0.013->100 0.025->100 0.025->100 0.2->100 0.2-> 100
0.39 0.78 0.2 0.39 1.56 1.56 0.78
6.25 >100 >100 >100 >100 >100 >100
0.025-0.1 0.1-0.78 0.025-0.2 0.1-0.78 0.05-0.78 0.2-0.78 0.2-0.39
0.05 0.39 0.1 0.2 0.39 0.39 0.2
0.1 0.39 0.1 0.39 0.39 0.78 0.39
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.013-12.5 0.1-100
0.025->100 0.05->100 0.05-> 100 0.2->100 0.1-> 100
0.05 0.2 0.05 0.2 0.39 0.2 0.2
12.5 100 >100 >100 >100 >100 100
Providencia rettgeri (25)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.025-6.25 0.1->100 0.025->100 0.1->100 0.1->100 0.2->100 0.1->100
1.56 12.5 25 6.25 6.25 25 12.5
6.25 >100 >100 25 >100 >100 >100
Providencia stuartii (25)
DU-6859 OFLX CPFX TFLX SPFX
0.1-6.25 0.78-100 0.1->100 0.1-25 0.2-25 0.39->100 0.2-50
0.2 1.56 0.78 0.39 0.39 1.56 0.78
3.13 25 50 12.5 6.25 50 50
0.013-6.25 0.1-100 0.013-50 0.05-6.25 0.1-50 0.1->100 0.1->100
0.025 0.2 0.05 0.2 0.2 0.2 0.1
0.2 1.56 0.78 1.56 1.56 3.13 1.56
0.05-0.78 0.39-3.13 0.05-0.78 0.1-0.78 0.2-3.13 0.39-6.25 0.2-6.25
0.2 1.56 0.39 0.39 0.78 1.56 1.56
0.39 3.13 0.39 0.78 1.56 3.13 3.13
0.39-50 6.25-> 100 0.39-> 100
3.13 25 3.13 6.25 12.5 50 25
DU-6859 OFLX CPFX TFLX
SPFX LFLX FLRX
Proteus mirabilis (50)
DU-6859
OFLX CPFX TFLX SPFX LFLX FLRX
Proteus vulgaris (50)
LFLX FLRX
Morganella morganii (50)
DU-6859 OFLX CPFX
TFLX SPFX LFLX FLRX
Pseudomonas aeruginosa
Quinolone susceptiblec (57)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
Quinolone resistantd (46)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.78->100 1.56->100 3.13->100
3.13-> 100
12.5 >100 50 >100 >100 >100 >100
Continued on following page
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ANTIMICROB. AGENTS CHEMOTHER. TABLE 1-Continued
Organism (no. of isolates)
Drug
MIC (jig/ml)
Range
50%
90%
Pseudomonas cepacia (25)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.1-3.13 0.78-25 0.78-25 0.39-25 0.2-25 1.56-50 0.78-25
1.56 12.5 3.13 6.25 3.13 12.5 6.25
3.13 25 12.5 25 25 50 25
Xanthomonas maltophilia (24)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.05-0.78 0.78-12.5 0.78-12.5 0.2-3.13 0.1-3.13 0.78-12.5 0.78-6.25
0.39 3.13 3.13 0.39 0.78 3.13 1.56
0.78 6.25 6.25 1.56 1.56 6.25 3.13
Haemophilus influenzae (44)
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
.0.003 0.05 0.013 0.006 .0.003 0.1 0.05
0.006 0.1 0.05 0.013 0.013 0.1 0.1
0.025 0.39 0.2 0.1 0.1 0.78 0.78 1.56
Neisseria gonorrhoeae (25)
100 >100
25 >100 >100 >100 >100 >100 >100
Continued on following page
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TABLE 1-Continued MIC (,ug/ml)
Drug
Organism (no. of isolates)
Range
Alcaligenes denitrificans subsp. xylosoxydans (25)
Bacteroides fiagillis (23)
50%
90%
3.13
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX
0.2-6.25
0.78
1.56-> 100 1.56-> 100 0.39-> 100 0.39-50 1.56->100 1.56-100
25 12.5 12.5 6.25 25 6.25
100 100 > 100 50 100 50
DU-6859 OFLX CPFX TFLX SPFX LFLX FLRX Clindamycin
0.05-0.78 1.56-50 3.13-100 0.39-12.5 0.78-25 6.25-50 6.25-100 0.05->100
0.1 3.13 6.25 0.78 1.56 12.5 12.5 0.2
12.5 50 1.56 1.56 25 25 > 100
0.39
a Methicillin susceptible, methicillin MIC of
