ANTIMICROBIAL AGENTS
AND
Vol. 35, No. 12
CHEMOTHERAPY, Dec. 1991, p. 2490-2495
0066-4804/91/122490-06$02.00/0 Copyright © 1991, American Society for Microbiology
In Vitro Antibacterial Activity of a New Quinolone, NM394 MASAKUNI OZAKI,l* MASATO MATSUDA,1 YOSHIFUMI TOMII,1 KIYOSHI KIMURA,' KENJI KAZUNO,2 MASAHIKO KITANO,2 MASAHIRO KISE,2 KAZUO SHIBATA,3 MASAKO OTSUKI,3 AND TAKESHI NISHINO3 Biology Laboratories' and Chemistry Laboratories,2 Nippon Shinyaku Co., Ltd., Nishiohji Hachijo, Minami-ku, Kyoto 601, and Department of Microbiology, Kyoto Pharmaceutical University,3 Kyoto 607, Japan Received 15 April 1991/Accepted 21 September 1991
NM394 is a new 6-fluoroquinolone antibacterial agent with a tricyclic structure which has a bridge that connects the N-1 and C-2 positions of the quinolone. The antibacterial activity of NM394 against clinical isolates of staphylococci, streptococci, enterococci, members of the family Enterobacteriaceae, and Pseudomonas aeruginosa was equal to or one-half that of ciprofloxacin. NM394 was as active as ofloxacin against gram-positive bacteria and was two to eight times more active against gram-negative bacteria, including P. aeruginosa. NM394 was two to eight times more active than enoxacin against gram-positive and gram-negative bacteria. The MICs of NM394 against Escherichia coil and P. aeruginosa at pH 5.5 were reduced 4 to 16 times compared with those at pH 7.0. Ciprofloxacin, ofloxacin, and enoxacin were 2 to 32 times less active against these two bacteria and Staphylococcus aureus at an acidic pH than they were at pH 7.0. In the presence of 5 mM Mg2+, the MICs of aUl of these drugs increased 2 to 32 times, but they were only slightly affected by 5 mM Ca2, type of medium, serum, or size of inoculum. NM394 showed potent bactericidal activity and inhibited the supercoiling activity of E. coli DNA gyrase. The in vitro antibacterial profile of NM394 is similar to that of other 6-fluoroquinolones.
NM394 is a new 6-fluoroquinolone with the chemical structure 6-fluoro-1-methyl-7-(1-piperazinyl)-4-oxo-4H-[1,3] thiazeto[3,2-a]quinoline-3-carboxylic acid (Fig. 1). It has broad antibacterial activity against gram-positive and gramnegative bacteria. 6-Fluoroquinolone derivatives such as norfloxacin (6), ofloxacin (15), enoxacin (9), and ciprofloxacin (17) have been reported to have significant antibacterial activities, but 6-fluoroquinolones with a substituent at the C-2 position have rarely been reported (2). We have tested quinolone derivatives with a substituted sulfur atom at the C-2 position and found 5-oxo-1,2-dihydro-5H-thiazolo[3,2a]quinoline carboxylic acid derivatives to have potent antibacterial activity (8). We also found that 4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline carboxylic acid derivatives have broader-spectrum and more potent antibacterial activities than those of 5-oxo-1,2-dihydro-5H-thiazolo[3,2-a]quinoline carboxylic acid derivatives, leading to the synthesis of NM394. In this study, we compared the in vitro antibacterial activity of NM394 with those of ciprofloxacin, ofloxacin, and enoxacin. (A part of this work was presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy
from 15 hospitals in Japan from 1986 to 1989 and that were maintained in the Department of Microbiology, Kyoto Pharmaceutical University, Kyoto, Japan. Susceptibility testing. MICs were determined by the agar dilution method recommended by the Japan Society of Chemotherapy (3). As the test medium, heart infusion agar (HIA; Nissui, Tokyo, Japan) supplemented with 5% horse blood was used for streptococci and Branhamella catarrhalis; HIA supplemented with 5% Fildes enrichment (Difco Laboratories, Detroit, Mich.) was used for Haemophilus influenzae. HIA was used for staphylococci, enterococci, and gram-negative enteric bacteria. Overnight cultures in trypto-soy broth (Nissui) at 37°C were diluted with the same broth, and 5 x 103 CFU per spot (5 ,ul) was inoculated onto the agar plates. All agar plates were incubated at 37°C for 18 h. Effects of various conditions on antibacterial activity. The effects of various conditions on the in vitro activities of NM394, ofloxacin, ciprofloxacin, and enoxacin against Staphylococcus aureus Smith, Escherichia coli KC-14, and Pseudomonas aeruginosa E-2 were determined. (i) Effect of medium. The effect of the medium on the MICs was determined with HIA, modified Mueller-Hinton agar (Eiken, Tokyo, Japan), trypto-soy agar (Nissui), and nutrient agar (Nissui). (ii) Effect of pH. The effect of pH on the MICs was determined on HIA adjusted to pH 5.5, 7.0, and 8.5. (iii) Effect of serum. The effect of serum on the MICs was
[11].)
MATERIALS AND METHODS Antimicrobial agents. NM394 was synthesized at the chemistry laboratories of Nippon Shinyaku Co., Ltd. The other drugs used in this study were ofloxacin (Daiichi Seiyaku Co., Ltd., Tokyo, Japan), ciprofloxacin (Bayer Yakuhin Co., Ltd., Osaka, Japan), enoxacin (Dainippon Seiyaku Co., Ltd., Osaka, Japan), and norfloxacin (Kyorin Seiyaku Co., Ltd. Tokyo, Japan). Bacterial strains. The bacterial strains used in this study were standard strains and clinical isolates that were obtained
HN-1ICH3
0 *
Corresponding author.
FIG. 1. Chemical structure of NM394. 2490
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ACTIVITY OF NM394
2491
TABLE 1. Antibacterial activities of NM394, ciprofloxacin, ofloxacin, and enoxacin against standard strains MIC (,ug/ml)
Organism
Staphylococcus aureus 209-P JC Staphylococcus aureus Smith Staphylococcus aureus Terajima Staphylococcus aureus Neumann Staphylococcus aureus E-46 Staphylococcus aureus No. 80 Micrococcus luteus ATCC 9341 Streptococcus pneumoniae type I Streptococcus pneumoniae type II Streptococcus pneumoniae type III Streptococcus pyogenes C-23 Streptococcus pyogenes Cook Streptococcus pyogenes C-203 Bacillus subtilis ATCC 6633 Corynebacterium diphtheriae Toronto Escherichia coli NIHJ JC-2 Escherichia coli NIH Escherichia coli K-12 Escherichia coli KC-14 Citrobacterfreundii NIH 10018-68 Salmonella typhi T-287 Salmonella typhi 0-901 Salmonella paratyphi A Salmonella paratyphi B Shigella dysenteriae EW-7 Shigella flexneri 2A EW-10 Shigella boydii EW-28 Shigella sonnei EW-33 Klebsiella pneumoniae KC-1 Klebsiella pneumoniae DT-S Klebsiella pneumoniae NCTC 9632 Enterobacter cloacae NCTC 9394 Enterobacter aerogenes NCTC 10006 Hafnia alvei NCTC 9540 Serratia marcescens IFO 3736 Serratia marcescens T-55 Proteus vulgaris OX-19 Proteus mirabilis 1287 Proteus mirabilis 181 Morganella morganii Kono Providencia rettgeri NIH 96 Pseudomonas aeruginosa No. 12 Pseudomonas aeruginosa NC-5 Pseudomonas aeruginosa E-2 Acinetobacter calcoaceticus Ac-54
NM394
Ciprofloxacin
Ofloxacin
Enoxacin
0.10 0.20 0.20 0.39 0.78 0.78 1.56 0.39 0.20 0.39 0.20 0.20 0.20 0.10 0.10 0.05 0.025 0.025 0.05 0.10 0.05 0.012 0.025 0.05 0.05 0.025 0.025 0.025 0.05 0.05 0.025 0.10 0.10 0.025 0.20 0.39 0.20 0.10 0.10 0.20 0.20 0.20 0.20 0.39 1.56
0.10 0.20 0.20 0.39 0.39 0.78 1.56 0.39 0.39 0.39 0.39 0.39 0.39 0.012 0.05 0.012 0. 006 0. 006 0.012 0.05 0.012 0. 006 0. 006 0.012 0.012 0.012 0.006 0. 006 0.012 0.025 0.012 0.025 0.05 -'0.006 0.10 0.10 0.10 0.025 0.025 0.025 0.10 0.20 0.025 0.20 0.78
0.39 0.20 0.39 0.20 0.39 0.39 1.56 0.78 0.78 0.78 0.78 0.78 0.78 0.05 0.10 0.10 0.025 0.05 0.05 0.20 0.025 0.012 0.05 0.05 0.10 0.05 0.05 0.05 0.05 0.05 0.05 0.10 0.10 0.05 0.39 0.20 0.05 0.10 0.20 0.20 0.05 0.78 0.20 0.78 0.39
0.78 0.78 0.78 0.78 0.78 1.56 12.5 3.13 1.56 3.13 3.13 1.56 3.13 0.20 0.39 0.20 0.10 0.10 0.20 0.20 0.20 0.10 0.10 0.20 0.20 0.20 0.20 0.10 0.20 0.20 0.20 0.39 0.39 0.10 0.39 0.39 0.20 0.20 0.39 0.39 0.10 0.78 0.20 1.56 3.13
determined with HIA supplemented with heat-inactivated horse serum to final concentrations of 10, 25, and 50%. (iv) Effects of cations. The effects of cations on the MICs were determined with HIA supplemented with 0.5 and 5 mM CaCl2, MgCl2, or NaCl. (v) Effect of inoculum size. The effect of inoculum size on the MICs was determined by inoculating 5 x 101 to 5 x 105 CFU per spot onto HIA plates. Bactericidal activity. The bactericidal activity of NM394 against S. aureus 209-P JC, E. coli KC-14, Serratia marcescens T-55, and P. aeruginosa E-2 was determined. An overnight culture in trypto-soy broth was diluted with fresh broth and incubated at 37°C with shaking to yield approximately 106 CFU/ml; then, NM394 was added to a final concentration of one-fourth to four times the MIC. After incubation for 1, 2, or 4 h at 37°C with shaking, samples (0.5 to 1 ml) were withdrawn and serially diluted. One milliliter of
the diluent was mixed with 15 ml of HIA and incubated at 37°C for 18 h, to count the CFU per milliliter. Frequency of spontaneous resistance. Mutants of S. aureus 143, S. aureus 56 (methicillin-resistant S. aureus), E. coli NIHJ JC-2, Enterobacter cloacae 8511, S. marcescens 4004, and P. aeruginosa OWPH 1625 that developed spontaneous resistance to NM394, ciprofloxacin, or ofloxacin were detected by plating 0.1 ml of an overnight culture in sensitivity test broth (Nissui), which was concentrated threefold by centrifugation, onto sensitivity test agar (Nissui) plates containing four times the MIC of each drug. After 48 h of incubation at 37°C, the colonies on the plates were counted. Inhibition of E. coli DNA gyrase. DNA gyrase subunits were purified by novobiocin Sepharose chromatography as described previously (4, 14, 16). The inhibitory effects of NM394, ciprofloxacin, ofloxacin, and norfloxacin against DNA gyrase supercoiling activity were measured by a
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ANTIMICROB. AGENTS CHEMOTHER.
OZAKI ET AL.
TABLE 2. Antibacterial activities of NM394, ciprofloxacin, ofloxacin, and enoxacin
Organism (no. or strains)
Staphylococcus aureus, methicillin susceptible (37)
MIC (Ig/ml)a
DrugRange
50
90%
1.56
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-3.13 0.39-6.25 0.20-1.56 0.78-12.5
0.78 0.39 0.39 1.56
1.56 3.13
Staphylococcus aureus, methicillin resistant (38)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.78-100 0.78-100 0.39-25 1.56-100
1.56 1.56 0.78 3.13
12.5 25 6.25 50
Staphylococcus epidermidis (35)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.10-1.56 0.20-0.78 0.39-0.78 0.78-6.25
0.78 0.39 0.78 1.56
0.78 0.39 0.78 1.56
Streptococcus pyogenes (34)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-1.56 0.39-1.56 0.78-1.56 3.13-12.5
0.78 0.39 0.78 3.13
0.78 0.78 1.56 12.5
Streptococcus pneumoniae (31)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-6.25 0.39-6.25 0.20-6.25 3.13-25
1.56 0.78 1.56 6.25
6.25 3.13 3.13 12.5
Enterococcus faecalis (40)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-3.13 0.39-1.56 0.78-3.13 3.13-25
1.56 0.78 1.56 6.25
1.56 1.56 3.13 12.5
Enterococcus faecium (30)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-12.5 0.20-12.5 0.78-12.5 3.13-25
1.56 0.78 3.13 25
3.13 3.13 6.25 25
Enterococcus avium (14)
NM394 Ciprofloxacin
1.56 0.78 1.56-3.13 6.25-12.5
1.56 0.78 1.56 6.25
1.56 0.78 3.13 12.5
Ofloxacin Enoxacin
1.56
Escherichia coli (43)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.05-0.39 0.025-0.39 0.10-0.78 0.39-3.13
0.10 0.05 0.10 0.39
0.10 0.05 0.20 0.39
Citrobacterfreundii (34)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.025-0.78 0.012-0.78 0.10-6.25 0.10-1.56
0.10 0.10 0.39 0.39
0.20 0.39 1.56 0.78
Enterobacter cloacae (42)
NM394
0.012-0.10
0.05 0.05 0.10 0.39
Enterobacter aerogenes (43)
Klebsiella pneumoniae (43)
Ciprofloxacin Ofloxacin Enoxacin
-'0.006-0.05 0.025-0.20 0.10-0.78
0.05 0.025 0.10 0.20
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.012-0.10 _0.006-0.10 0.05-0.20 0.05-0.39
0.05 0.05 0.10 0.20
0.05 0.05 0.20 0.39
NM394
0.05-0.78 '0.006-0.39 0.10-1.56 c0.006-3.13
0.10 0.10 0.20 0.78
0.39 0.39 0.78 1.56
0.10-1.56 0.05-0.39 0.20-1.56 0.39-3.13
0.20 0.10 0.20 0.78
0.78 0.39 0.39 1.56
Ciprofloxacin Ofloxacin Enoxacin Proteus mirabilis (41)
NM394
Ciprofloxacin Ofloxacin Enoxacin
Continued on following page
VOL. 35, 1991
ACTIVITY OF NM394
2493
TABLE 2-Continued Organism
MIC (Iug/ml)a
DrugRange
(no. of strains)
50
90
Proteus vulgaris (43)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.05-0.39 0.025-0.20 0.10-0.78 0.20-1.56
0.10 0.05 0.20 0.39
0.20 0.10 0.39 0.78
Morganella morganii (42)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.05-3.13 0.012-1.56 0.025-6.25 0.10-6.25
0.10 0.05 0.20 0.39
0.78 0.39 0.78 1.56
Providencia rettgeri (25)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.025-3.13 0.025-1.56 0.10-6.25 0.20-6.25
0.20 0.10 0.39 0.39
0.78 0.78 6.25 6.25
Serratia marcescens (43)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.10-6.25 0.10-12.5 0.20-12.5 0.20-50
0.39 0.39 1.56 3.13
6.25 6.25 12.5 25
Pseudomonas aeruginosa (42)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.20-1.56 0.10-1.56 0.78-6.25 0.78-6.25
0.39 0.20 1.56 1.56
0.78 0.39 3.13 3.13
NM394
0.20-12.5 0.10-12.5
3.13 0.78 0.78 6.25
Acinetobacter calcoaceticus (40)
Haemophilus influenzae (48)
Branhamella catarrhalis (10)
Ciprofloxacin Ofloxacin Enoxacin
0.10-12.5 0.78-50
1.56 0.20 0.39 3.13
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.012-0.10 .0 006-0.05 0.025-0.39 0.10-0.78
0.025 0.012 0.025 0.10
0.05 0.025 0.05 0.20
0.20 0.10 0.10 0.20
0.20 0.10 0.20 0.20
NM394
Ciprofloxacin Ofloxacin Enoxacin
0.10-0.20 0.05-0.10 0.10-0.20 0.20-0.39
a 50%o and 90%o, MICs for 50 and 90%o of isolates tested, respectively.
modification of the method described in previous reports (4, 5). The mixture containing subunits A and B (one unit each; one unit of enzyme was defined as the amount that brought 50% of relaxed pBR322 to the supercoiled form), an appro-
TABLE 3. Effect of medium on antibacterial activity MIC (pg/ml)b
Organism
Mediuma
Staphylococcus
HIA STA TSA NA
0.20 0.20 0.20 0.10
0.10 0.10 0.20 0.10
0.20 0.20 0.20 0.20
0.78 0.78 0.78 0.39
HIA STA TSA NA
0.025 0.025 0.025 0.025
0.012 0.012 0.025 0.012
0.05 0.05 0.10 0.05
0.20 0.20 0.20 0.20
HIA STA TSA NA
0.20 0.20 0.20 0.20
0.20 0.20 0.39 0.20
0.78 1.56 1.56 0.78
0.78
aureus
NM394
Smith
Escherichia coli KC-14
Pseudomonas
aeruginosa E-2
C
Cpro Ofloxacin Enoxacin floxacin
0.78 1.56 0.78
a HIA, heart infusion agar; STA, modified Mueller-Hinton agar; TSA,
trypto-soy agar; NA, nutrient agar. b Inoculum size, 5
x
103 CFU (5
Rl) per spot.
TABLE 4. Effect of medium pH on antibacterial activity MIC (pLg/ml)a
Organism
pH NM394
Staphylococcus aureus 5.0 0.20 Smith 7.0 0.20
Cpo Ofloxacin Enoxacin CiProfloxacin
0.39 0.20 0.20
0.78 0.20 0.20
1.56 0.39 0.39
Escherichia coli KC-14 5.5 0.39 7.0 0.025 8.5 0.025
0.39 0.012 0.012
0.78 0.10 0.10
1.56 0.10 0.10
Pseudomonas aeruginosa E-2
1.56 0.20 0.20
6.25 1.56 1.56
6.25 0.78 0.78
8.5
5.5 7.0 8.5
0.20
0.78 0.20 0.20
a In heart infusion agar; inoculum size, 5 x 103 CFU (5
p.d) per spot.
2494
OZAKI ET AL.
10]
A
ANTIMICROB. AGENTS CHEMOTHER. TABLE 6. Inhibition of supercoiling activity of E. coli K-12 DNA gyrase by NM394 and other quinolones
control
0.05 n
o
~~~o
0
0.78
1
4
2
Time (hO
rrme 10
E
control
~~~~~~0.10*
M -
039
0.20
I
0.39 0.78
~~~~~~~~~1.5
1.5 1
2
MIC
NM394 0.84 Ciprofloxacin 0.69 Ofloxacin 2.25 Norfloxacin 1.65 a ICW, 50% inhibitory concentration.
0.012 0.012 0.10 0.10
(h>)
D
E
gm
I~(g/C
trug
0.20 0.39
4
1
Time (hr)
2
4
Time
(hr)
FIG. 2. Bactericidal activity of NM394 against S. aureus 209-P JC (MIC, 0.20 ,ug/ml) (A), E. coli KC-14 (MIC, 0.025 ,tg/ml) (B), S. marcescens T-55 (MIC, 0.39 pLg/ml) (C), and P. aeruginosa E-2 (MIC, 0.39 jg/ml) (D). The asterisk indicates micrograms per nilliliter.
priate concentration of the drug, and relaxed pBR322 were incubated at 30°C for 1 h; then, phenol-chloroform (1:1) was added to the reaction mixture. The DNA that was recovered from the water layer was placed in a 0.8% agarose gel and electrophoresed. The gel was stained with ethidium bromide (0.5 pug/ml). The DNA-containing bands under UV light (Transilluminator; Funakoshi, Tokyo, Japan) were photographed. The densitometric analysis of the negative film was done with a dual-wavelength thin-layer chromatographic scanner (Shimadzu, Kyoto, Japan). The 50% inhibitory concentrations were defined as the concentrations of the drugs that inhibited the supercoiling activity of gyrase by 50%. RESULTS
Susceptibility tests. The antibacterial activities of NM394, ciprofloxacin, ofloxacin, and enoxacin against 54 standard strains and 22 species and against 10 to 48 strains of clinical isolates are given in Tables 1 and 2. The antibacterial activity
of NM394 against standard strains was equal to or one-half that of ciprofloxacin. The MICs of NM394 for 90%6 of clinically isolated staphylococci, streptococci, and enterococci ranged from 0.78 to 12.5 pug/ml. The activity of NM394 against enterococci was equal to or one-half that of ciprofloxacin, but it was twice that of ofloxacin. NM394 was as active as ciprofloxacin and ofloxacin against S. aureus and other gram-positive bacteria. NM394 was 2 to 16 times more active than enoxacin against gram-positive bacteria. The MICs of NM394 for 90% of isolates tested ranged from 0.05 to 6.25 p,g/ml for members of the family Enterobacteriaceae and P. aeruginosa. The activity of NM394 was equal to or one-half that of ciprofloxacin, but it was two to eight times that of ofloxacin and enoxacin. Effect of various conditions on antibacterial activity. The MICs of NM394, ciprofloxacin, ofloxacin, and enoxacin determined in modified Mueller-Hinton agar, trypto-soy agar, and nutrient agar were equal to or almost equal to those in HIA (Table 3). The MIC of NM394 for S. aureus Smith was the same at pH 5.5, 7.0, and 8.5, but it was 16-fold higher for E. coli KC-14 and 4-fold higher for P. aeruginosa E-2 at pH 5.5 as it was at pH 7.0. The MICs of ciprofloxacin, ofioxacin, and enoxacin for these three bacteria were 2- to 32-fold higher at pH 5.5 than they were at pH 7.0 (Table 4). The MICs of NM394, ciprofloxacin, ofloxacin, and enoxacin were affected slightly at a concentration of 10 to 50% serum or an inoculum of 5 x 101 to 5 x 10' CFU per spot. The MICs of NM394, ciprofloxacin, ofloxacin, and enoxacin were not affected by 5 mM Ca2+, 5 mM Na+, or 0.5 mM Mg2"; but they were increased 4- to 16-fold in the presence of 5 mM Mg2+. Bactericidal effect. The bactericidal activity of NM394 against S. aureus 209-P JC, E. coli KC-14, S. marcescens T-55, and P. aeruginosa E-2 is shown in Fig. 2. Viable cells of S. aureus 209-P JC were reduced by 1 to 3 log1o CFU/ml within 4 h after exposure to one to four times the MIC of NM394. After exposure to the same concentration of NM394, viable cells of E. coli KC-14, S. marcescens T-55,
TABLE 5. Frequency of development of spontaneous resistancea Organism
Staphylococcus aureus 143b Staphylococcus aureus 56C Escherichia coli NIHJ JC-2 Enterobacter cloacae 8511"
Serratia marcescens 4014b Pseudomonas aeruginosa OWPH 1625b a Selected at four times the MICs. b Clinical isolates. c Clinical isolate, methicillin resistant.
Frequency of development of spontaneous resistance NM394
AND
Vol. 35, No. 12
CHEMOTHERAPY, Dec. 1991, p. 2490-2495
0066-4804/91/122490-06$02.00/0 Copyright © 1991, American Society for Microbiology
In Vitro Antibacterial Activity of a New Quinolone, NM394 MASAKUNI OZAKI,l* MASATO MATSUDA,1 YOSHIFUMI TOMII,1 KIYOSHI KIMURA,' KENJI KAZUNO,2 MASAHIKO KITANO,2 MASAHIRO KISE,2 KAZUO SHIBATA,3 MASAKO OTSUKI,3 AND TAKESHI NISHINO3 Biology Laboratories' and Chemistry Laboratories,2 Nippon Shinyaku Co., Ltd., Nishiohji Hachijo, Minami-ku, Kyoto 601, and Department of Microbiology, Kyoto Pharmaceutical University,3 Kyoto 607, Japan Received 15 April 1991/Accepted 21 September 1991
NM394 is a new 6-fluoroquinolone antibacterial agent with a tricyclic structure which has a bridge that connects the N-1 and C-2 positions of the quinolone. The antibacterial activity of NM394 against clinical isolates of staphylococci, streptococci, enterococci, members of the family Enterobacteriaceae, and Pseudomonas aeruginosa was equal to or one-half that of ciprofloxacin. NM394 was as active as ofloxacin against gram-positive bacteria and was two to eight times more active against gram-negative bacteria, including P. aeruginosa. NM394 was two to eight times more active than enoxacin against gram-positive and gram-negative bacteria. The MICs of NM394 against Escherichia coil and P. aeruginosa at pH 5.5 were reduced 4 to 16 times compared with those at pH 7.0. Ciprofloxacin, ofloxacin, and enoxacin were 2 to 32 times less active against these two bacteria and Staphylococcus aureus at an acidic pH than they were at pH 7.0. In the presence of 5 mM Mg2+, the MICs of aUl of these drugs increased 2 to 32 times, but they were only slightly affected by 5 mM Ca2, type of medium, serum, or size of inoculum. NM394 showed potent bactericidal activity and inhibited the supercoiling activity of E. coli DNA gyrase. The in vitro antibacterial profile of NM394 is similar to that of other 6-fluoroquinolones.
NM394 is a new 6-fluoroquinolone with the chemical structure 6-fluoro-1-methyl-7-(1-piperazinyl)-4-oxo-4H-[1,3] thiazeto[3,2-a]quinoline-3-carboxylic acid (Fig. 1). It has broad antibacterial activity against gram-positive and gramnegative bacteria. 6-Fluoroquinolone derivatives such as norfloxacin (6), ofloxacin (15), enoxacin (9), and ciprofloxacin (17) have been reported to have significant antibacterial activities, but 6-fluoroquinolones with a substituent at the C-2 position have rarely been reported (2). We have tested quinolone derivatives with a substituted sulfur atom at the C-2 position and found 5-oxo-1,2-dihydro-5H-thiazolo[3,2a]quinoline carboxylic acid derivatives to have potent antibacterial activity (8). We also found that 4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline carboxylic acid derivatives have broader-spectrum and more potent antibacterial activities than those of 5-oxo-1,2-dihydro-5H-thiazolo[3,2-a]quinoline carboxylic acid derivatives, leading to the synthesis of NM394. In this study, we compared the in vitro antibacterial activity of NM394 with those of ciprofloxacin, ofloxacin, and enoxacin. (A part of this work was presented at the 29th Interscience Conference on Antimicrobial Agents and Chemotherapy
from 15 hospitals in Japan from 1986 to 1989 and that were maintained in the Department of Microbiology, Kyoto Pharmaceutical University, Kyoto, Japan. Susceptibility testing. MICs were determined by the agar dilution method recommended by the Japan Society of Chemotherapy (3). As the test medium, heart infusion agar (HIA; Nissui, Tokyo, Japan) supplemented with 5% horse blood was used for streptococci and Branhamella catarrhalis; HIA supplemented with 5% Fildes enrichment (Difco Laboratories, Detroit, Mich.) was used for Haemophilus influenzae. HIA was used for staphylococci, enterococci, and gram-negative enteric bacteria. Overnight cultures in trypto-soy broth (Nissui) at 37°C were diluted with the same broth, and 5 x 103 CFU per spot (5 ,ul) was inoculated onto the agar plates. All agar plates were incubated at 37°C for 18 h. Effects of various conditions on antibacterial activity. The effects of various conditions on the in vitro activities of NM394, ofloxacin, ciprofloxacin, and enoxacin against Staphylococcus aureus Smith, Escherichia coli KC-14, and Pseudomonas aeruginosa E-2 were determined. (i) Effect of medium. The effect of the medium on the MICs was determined with HIA, modified Mueller-Hinton agar (Eiken, Tokyo, Japan), trypto-soy agar (Nissui), and nutrient agar (Nissui). (ii) Effect of pH. The effect of pH on the MICs was determined on HIA adjusted to pH 5.5, 7.0, and 8.5. (iii) Effect of serum. The effect of serum on the MICs was
[11].)
MATERIALS AND METHODS Antimicrobial agents. NM394 was synthesized at the chemistry laboratories of Nippon Shinyaku Co., Ltd. The other drugs used in this study were ofloxacin (Daiichi Seiyaku Co., Ltd., Tokyo, Japan), ciprofloxacin (Bayer Yakuhin Co., Ltd., Osaka, Japan), enoxacin (Dainippon Seiyaku Co., Ltd., Osaka, Japan), and norfloxacin (Kyorin Seiyaku Co., Ltd. Tokyo, Japan). Bacterial strains. The bacterial strains used in this study were standard strains and clinical isolates that were obtained
HN-1ICH3
0 *
Corresponding author.
FIG. 1. Chemical structure of NM394. 2490
VOL. 35, 1991
ACTIVITY OF NM394
2491
TABLE 1. Antibacterial activities of NM394, ciprofloxacin, ofloxacin, and enoxacin against standard strains MIC (,ug/ml)
Organism
Staphylococcus aureus 209-P JC Staphylococcus aureus Smith Staphylococcus aureus Terajima Staphylococcus aureus Neumann Staphylococcus aureus E-46 Staphylococcus aureus No. 80 Micrococcus luteus ATCC 9341 Streptococcus pneumoniae type I Streptococcus pneumoniae type II Streptococcus pneumoniae type III Streptococcus pyogenes C-23 Streptococcus pyogenes Cook Streptococcus pyogenes C-203 Bacillus subtilis ATCC 6633 Corynebacterium diphtheriae Toronto Escherichia coli NIHJ JC-2 Escherichia coli NIH Escherichia coli K-12 Escherichia coli KC-14 Citrobacterfreundii NIH 10018-68 Salmonella typhi T-287 Salmonella typhi 0-901 Salmonella paratyphi A Salmonella paratyphi B Shigella dysenteriae EW-7 Shigella flexneri 2A EW-10 Shigella boydii EW-28 Shigella sonnei EW-33 Klebsiella pneumoniae KC-1 Klebsiella pneumoniae DT-S Klebsiella pneumoniae NCTC 9632 Enterobacter cloacae NCTC 9394 Enterobacter aerogenes NCTC 10006 Hafnia alvei NCTC 9540 Serratia marcescens IFO 3736 Serratia marcescens T-55 Proteus vulgaris OX-19 Proteus mirabilis 1287 Proteus mirabilis 181 Morganella morganii Kono Providencia rettgeri NIH 96 Pseudomonas aeruginosa No. 12 Pseudomonas aeruginosa NC-5 Pseudomonas aeruginosa E-2 Acinetobacter calcoaceticus Ac-54
NM394
Ciprofloxacin
Ofloxacin
Enoxacin
0.10 0.20 0.20 0.39 0.78 0.78 1.56 0.39 0.20 0.39 0.20 0.20 0.20 0.10 0.10 0.05 0.025 0.025 0.05 0.10 0.05 0.012 0.025 0.05 0.05 0.025 0.025 0.025 0.05 0.05 0.025 0.10 0.10 0.025 0.20 0.39 0.20 0.10 0.10 0.20 0.20 0.20 0.20 0.39 1.56
0.10 0.20 0.20 0.39 0.39 0.78 1.56 0.39 0.39 0.39 0.39 0.39 0.39 0.012 0.05 0.012 0. 006 0. 006 0.012 0.05 0.012 0. 006 0. 006 0.012 0.012 0.012 0.006 0. 006 0.012 0.025 0.012 0.025 0.05 -'0.006 0.10 0.10 0.10 0.025 0.025 0.025 0.10 0.20 0.025 0.20 0.78
0.39 0.20 0.39 0.20 0.39 0.39 1.56 0.78 0.78 0.78 0.78 0.78 0.78 0.05 0.10 0.10 0.025 0.05 0.05 0.20 0.025 0.012 0.05 0.05 0.10 0.05 0.05 0.05 0.05 0.05 0.05 0.10 0.10 0.05 0.39 0.20 0.05 0.10 0.20 0.20 0.05 0.78 0.20 0.78 0.39
0.78 0.78 0.78 0.78 0.78 1.56 12.5 3.13 1.56 3.13 3.13 1.56 3.13 0.20 0.39 0.20 0.10 0.10 0.20 0.20 0.20 0.10 0.10 0.20 0.20 0.20 0.20 0.10 0.20 0.20 0.20 0.39 0.39 0.10 0.39 0.39 0.20 0.20 0.39 0.39 0.10 0.78 0.20 1.56 3.13
determined with HIA supplemented with heat-inactivated horse serum to final concentrations of 10, 25, and 50%. (iv) Effects of cations. The effects of cations on the MICs were determined with HIA supplemented with 0.5 and 5 mM CaCl2, MgCl2, or NaCl. (v) Effect of inoculum size. The effect of inoculum size on the MICs was determined by inoculating 5 x 101 to 5 x 105 CFU per spot onto HIA plates. Bactericidal activity. The bactericidal activity of NM394 against S. aureus 209-P JC, E. coli KC-14, Serratia marcescens T-55, and P. aeruginosa E-2 was determined. An overnight culture in trypto-soy broth was diluted with fresh broth and incubated at 37°C with shaking to yield approximately 106 CFU/ml; then, NM394 was added to a final concentration of one-fourth to four times the MIC. After incubation for 1, 2, or 4 h at 37°C with shaking, samples (0.5 to 1 ml) were withdrawn and serially diluted. One milliliter of
the diluent was mixed with 15 ml of HIA and incubated at 37°C for 18 h, to count the CFU per milliliter. Frequency of spontaneous resistance. Mutants of S. aureus 143, S. aureus 56 (methicillin-resistant S. aureus), E. coli NIHJ JC-2, Enterobacter cloacae 8511, S. marcescens 4004, and P. aeruginosa OWPH 1625 that developed spontaneous resistance to NM394, ciprofloxacin, or ofloxacin were detected by plating 0.1 ml of an overnight culture in sensitivity test broth (Nissui), which was concentrated threefold by centrifugation, onto sensitivity test agar (Nissui) plates containing four times the MIC of each drug. After 48 h of incubation at 37°C, the colonies on the plates were counted. Inhibition of E. coli DNA gyrase. DNA gyrase subunits were purified by novobiocin Sepharose chromatography as described previously (4, 14, 16). The inhibitory effects of NM394, ciprofloxacin, ofloxacin, and norfloxacin against DNA gyrase supercoiling activity were measured by a
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ANTIMICROB. AGENTS CHEMOTHER.
OZAKI ET AL.
TABLE 2. Antibacterial activities of NM394, ciprofloxacin, ofloxacin, and enoxacin
Organism (no. or strains)
Staphylococcus aureus, methicillin susceptible (37)
MIC (Ig/ml)a
DrugRange
50
90%
1.56
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-3.13 0.39-6.25 0.20-1.56 0.78-12.5
0.78 0.39 0.39 1.56
1.56 3.13
Staphylococcus aureus, methicillin resistant (38)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.78-100 0.78-100 0.39-25 1.56-100
1.56 1.56 0.78 3.13
12.5 25 6.25 50
Staphylococcus epidermidis (35)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.10-1.56 0.20-0.78 0.39-0.78 0.78-6.25
0.78 0.39 0.78 1.56
0.78 0.39 0.78 1.56
Streptococcus pyogenes (34)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-1.56 0.39-1.56 0.78-1.56 3.13-12.5
0.78 0.39 0.78 3.13
0.78 0.78 1.56 12.5
Streptococcus pneumoniae (31)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-6.25 0.39-6.25 0.20-6.25 3.13-25
1.56 0.78 1.56 6.25
6.25 3.13 3.13 12.5
Enterococcus faecalis (40)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-3.13 0.39-1.56 0.78-3.13 3.13-25
1.56 0.78 1.56 6.25
1.56 1.56 3.13 12.5
Enterococcus faecium (30)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.39-12.5 0.20-12.5 0.78-12.5 3.13-25
1.56 0.78 3.13 25
3.13 3.13 6.25 25
Enterococcus avium (14)
NM394 Ciprofloxacin
1.56 0.78 1.56-3.13 6.25-12.5
1.56 0.78 1.56 6.25
1.56 0.78 3.13 12.5
Ofloxacin Enoxacin
1.56
Escherichia coli (43)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.05-0.39 0.025-0.39 0.10-0.78 0.39-3.13
0.10 0.05 0.10 0.39
0.10 0.05 0.20 0.39
Citrobacterfreundii (34)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.025-0.78 0.012-0.78 0.10-6.25 0.10-1.56
0.10 0.10 0.39 0.39
0.20 0.39 1.56 0.78
Enterobacter cloacae (42)
NM394
0.012-0.10
0.05 0.05 0.10 0.39
Enterobacter aerogenes (43)
Klebsiella pneumoniae (43)
Ciprofloxacin Ofloxacin Enoxacin
-'0.006-0.05 0.025-0.20 0.10-0.78
0.05 0.025 0.10 0.20
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.012-0.10 _0.006-0.10 0.05-0.20 0.05-0.39
0.05 0.05 0.10 0.20
0.05 0.05 0.20 0.39
NM394
0.05-0.78 '0.006-0.39 0.10-1.56 c0.006-3.13
0.10 0.10 0.20 0.78
0.39 0.39 0.78 1.56
0.10-1.56 0.05-0.39 0.20-1.56 0.39-3.13
0.20 0.10 0.20 0.78
0.78 0.39 0.39 1.56
Ciprofloxacin Ofloxacin Enoxacin Proteus mirabilis (41)
NM394
Ciprofloxacin Ofloxacin Enoxacin
Continued on following page
VOL. 35, 1991
ACTIVITY OF NM394
2493
TABLE 2-Continued Organism
MIC (Iug/ml)a
DrugRange
(no. of strains)
50
90
Proteus vulgaris (43)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.05-0.39 0.025-0.20 0.10-0.78 0.20-1.56
0.10 0.05 0.20 0.39
0.20 0.10 0.39 0.78
Morganella morganii (42)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.05-3.13 0.012-1.56 0.025-6.25 0.10-6.25
0.10 0.05 0.20 0.39
0.78 0.39 0.78 1.56
Providencia rettgeri (25)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.025-3.13 0.025-1.56 0.10-6.25 0.20-6.25
0.20 0.10 0.39 0.39
0.78 0.78 6.25 6.25
Serratia marcescens (43)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.10-6.25 0.10-12.5 0.20-12.5 0.20-50
0.39 0.39 1.56 3.13
6.25 6.25 12.5 25
Pseudomonas aeruginosa (42)
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.20-1.56 0.10-1.56 0.78-6.25 0.78-6.25
0.39 0.20 1.56 1.56
0.78 0.39 3.13 3.13
NM394
0.20-12.5 0.10-12.5
3.13 0.78 0.78 6.25
Acinetobacter calcoaceticus (40)
Haemophilus influenzae (48)
Branhamella catarrhalis (10)
Ciprofloxacin Ofloxacin Enoxacin
0.10-12.5 0.78-50
1.56 0.20 0.39 3.13
NM394 Ciprofloxacin Ofloxacin Enoxacin
0.012-0.10 .0 006-0.05 0.025-0.39 0.10-0.78
0.025 0.012 0.025 0.10
0.05 0.025 0.05 0.20
0.20 0.10 0.10 0.20
0.20 0.10 0.20 0.20
NM394
Ciprofloxacin Ofloxacin Enoxacin
0.10-0.20 0.05-0.10 0.10-0.20 0.20-0.39
a 50%o and 90%o, MICs for 50 and 90%o of isolates tested, respectively.
modification of the method described in previous reports (4, 5). The mixture containing subunits A and B (one unit each; one unit of enzyme was defined as the amount that brought 50% of relaxed pBR322 to the supercoiled form), an appro-
TABLE 3. Effect of medium on antibacterial activity MIC (pg/ml)b
Organism
Mediuma
Staphylococcus
HIA STA TSA NA
0.20 0.20 0.20 0.10
0.10 0.10 0.20 0.10
0.20 0.20 0.20 0.20
0.78 0.78 0.78 0.39
HIA STA TSA NA
0.025 0.025 0.025 0.025
0.012 0.012 0.025 0.012
0.05 0.05 0.10 0.05
0.20 0.20 0.20 0.20
HIA STA TSA NA
0.20 0.20 0.20 0.20
0.20 0.20 0.39 0.20
0.78 1.56 1.56 0.78
0.78
aureus
NM394
Smith
Escherichia coli KC-14
Pseudomonas
aeruginosa E-2
C
Cpro Ofloxacin Enoxacin floxacin
0.78 1.56 0.78
a HIA, heart infusion agar; STA, modified Mueller-Hinton agar; TSA,
trypto-soy agar; NA, nutrient agar. b Inoculum size, 5
x
103 CFU (5
Rl) per spot.
TABLE 4. Effect of medium pH on antibacterial activity MIC (pLg/ml)a
Organism
pH NM394
Staphylococcus aureus 5.0 0.20 Smith 7.0 0.20
Cpo Ofloxacin Enoxacin CiProfloxacin
0.39 0.20 0.20
0.78 0.20 0.20
1.56 0.39 0.39
Escherichia coli KC-14 5.5 0.39 7.0 0.025 8.5 0.025
0.39 0.012 0.012
0.78 0.10 0.10
1.56 0.10 0.10
Pseudomonas aeruginosa E-2
1.56 0.20 0.20
6.25 1.56 1.56
6.25 0.78 0.78
8.5
5.5 7.0 8.5
0.20
0.78 0.20 0.20
a In heart infusion agar; inoculum size, 5 x 103 CFU (5
p.d) per spot.
2494
OZAKI ET AL.
10]
A
ANTIMICROB. AGENTS CHEMOTHER. TABLE 6. Inhibition of supercoiling activity of E. coli K-12 DNA gyrase by NM394 and other quinolones
control
0.05 n
o
~~~o
0
0.78
1
4
2
Time (hO
rrme 10
E
control
~~~~~~0.10*
M -
039
0.20
I
0.39 0.78
~~~~~~~~~1.5
1.5 1
2
MIC
NM394 0.84 Ciprofloxacin 0.69 Ofloxacin 2.25 Norfloxacin 1.65 a ICW, 50% inhibitory concentration.
0.012 0.012 0.10 0.10
(h>)
D
E
gm
I~(g/C
trug
0.20 0.39
4
1
Time (hr)
2
4
Time
(hr)
FIG. 2. Bactericidal activity of NM394 against S. aureus 209-P JC (MIC, 0.20 ,ug/ml) (A), E. coli KC-14 (MIC, 0.025 ,tg/ml) (B), S. marcescens T-55 (MIC, 0.39 pLg/ml) (C), and P. aeruginosa E-2 (MIC, 0.39 jg/ml) (D). The asterisk indicates micrograms per nilliliter.
priate concentration of the drug, and relaxed pBR322 were incubated at 30°C for 1 h; then, phenol-chloroform (1:1) was added to the reaction mixture. The DNA that was recovered from the water layer was placed in a 0.8% agarose gel and electrophoresed. The gel was stained with ethidium bromide (0.5 pug/ml). The DNA-containing bands under UV light (Transilluminator; Funakoshi, Tokyo, Japan) were photographed. The densitometric analysis of the negative film was done with a dual-wavelength thin-layer chromatographic scanner (Shimadzu, Kyoto, Japan). The 50% inhibitory concentrations were defined as the concentrations of the drugs that inhibited the supercoiling activity of gyrase by 50%. RESULTS
Susceptibility tests. The antibacterial activities of NM394, ciprofloxacin, ofloxacin, and enoxacin against 54 standard strains and 22 species and against 10 to 48 strains of clinical isolates are given in Tables 1 and 2. The antibacterial activity
of NM394 against standard strains was equal to or one-half that of ciprofloxacin. The MICs of NM394 for 90%6 of clinically isolated staphylococci, streptococci, and enterococci ranged from 0.78 to 12.5 pug/ml. The activity of NM394 against enterococci was equal to or one-half that of ciprofloxacin, but it was twice that of ofloxacin. NM394 was as active as ciprofloxacin and ofloxacin against S. aureus and other gram-positive bacteria. NM394 was 2 to 16 times more active than enoxacin against gram-positive bacteria. The MICs of NM394 for 90% of isolates tested ranged from 0.05 to 6.25 p,g/ml for members of the family Enterobacteriaceae and P. aeruginosa. The activity of NM394 was equal to or one-half that of ciprofloxacin, but it was two to eight times that of ofloxacin and enoxacin. Effect of various conditions on antibacterial activity. The MICs of NM394, ciprofloxacin, ofloxacin, and enoxacin determined in modified Mueller-Hinton agar, trypto-soy agar, and nutrient agar were equal to or almost equal to those in HIA (Table 3). The MIC of NM394 for S. aureus Smith was the same at pH 5.5, 7.0, and 8.5, but it was 16-fold higher for E. coli KC-14 and 4-fold higher for P. aeruginosa E-2 at pH 5.5 as it was at pH 7.0. The MICs of ciprofloxacin, ofioxacin, and enoxacin for these three bacteria were 2- to 32-fold higher at pH 5.5 than they were at pH 7.0 (Table 4). The MICs of NM394, ciprofloxacin, ofloxacin, and enoxacin were affected slightly at a concentration of 10 to 50% serum or an inoculum of 5 x 101 to 5 x 10' CFU per spot. The MICs of NM394, ciprofloxacin, ofloxacin, and enoxacin were not affected by 5 mM Ca2+, 5 mM Na+, or 0.5 mM Mg2"; but they were increased 4- to 16-fold in the presence of 5 mM Mg2+. Bactericidal effect. The bactericidal activity of NM394 against S. aureus 209-P JC, E. coli KC-14, S. marcescens T-55, and P. aeruginosa E-2 is shown in Fig. 2. Viable cells of S. aureus 209-P JC were reduced by 1 to 3 log1o CFU/ml within 4 h after exposure to one to four times the MIC of NM394. After exposure to the same concentration of NM394, viable cells of E. coli KC-14, S. marcescens T-55,
TABLE 5. Frequency of development of spontaneous resistancea Organism
Staphylococcus aureus 143b Staphylococcus aureus 56C Escherichia coli NIHJ JC-2 Enterobacter cloacae 8511"
Serratia marcescens 4014b Pseudomonas aeruginosa OWPH 1625b a Selected at four times the MICs. b Clinical isolates. c Clinical isolate, methicillin resistant.
Frequency of development of spontaneous resistance NM394
