Journal of Antimicrobial Chemotherapy (1996) 37, 1091-1102
Effect of growth conditions on antimicrobial activity of DU-6859a and its
bactericidal activity determined by the killing curve method
Mayumi Tanaka', Kazuki Hoshino*, Masato Hohmura*, Hiroko Ishida",
Akihiro Kitamura', Kenichi Sato', Isao Hayakawa* and Takeshi Nishino'
"New Product Research Laboratories I, Daiichi Pharmaceutical Co, Ltd., 16-13,
Kita-kasai 1-chome, Edogawa-ku, Tokyo 134, and
b
Department of Microbiology, Kyoto Pharmaceutical University, 5 Nakauchicho,
Misasagi, Yamashina-ku, Kyoto 607, Japan
The effect of growth conditions on the antibacterial activity of DU-6859a against
Staphylococcus auerus, Escherichia coli, and Pseudomonas aeruginosa was compared
with those of levofloxacin, sparfloxacin, and ciprofloxacin. This activity was not
affected by different media, inoculum size or the addition of human serum, but was
decreased under acidic conditions, in human urine, and in the presence of magnesium
and ferrous ion, as were the other quinolones tested. Time-kill curve studies
demonstrated the bactericidal action of DU-6859a against S. aureus, Streptococcus
pneumoniae, E. coli, and P. aeruginosa. Morphological alteration of these bacteria
after exposure to DU-6859a also demonstrated its bactericidal activity. The
frequency of spontaneous resistance to DU-6859a was less than or equal to those
of the reference drugs.
Introduction
DU-6859a, a novel quinolone antibacterial agent, has activity against a wide range of
bacteria and is particularly effective against Gram-positive bacteria (Sato et al., 1992).
The activity of quinolones is known to be decreased under acidic conditions and in the
presence of magnesium and aluminum ions (Smith, Eng & Cherubin, 1988; Smith, 1990;
Canton et al., 1992). Some antacids containing magnesium and aluminum ions have
been reported to antagonize the actions of quinolones (Shiba et al., 1992; Jaehde et al.,
1994). The present study investigated the effect of growth conditions, including different
media, inoculum size and medium pH, urine, and the addition of metal ions and human
serum, on the activity of DU-6859a. We also studied the bactericidal activity of
DU-6859a. The standard MIC method for determining antibacterial activity provides
no information on initial killing kinetics. This study therefore included the killing curve
method to obtain additional information on the antibacterial properties of DU-6859a
in vitro, particularly with regard to its initial bactericidal activity against selected strains.
The morphological study using electron microscopy also provides additional
information on its bactericidal activity and mode of action.
0305-7453/96/061091 + 12 $12.00/0
1091
© 1996 The British Society for Antimicrobial Chemotherapy
M. Tanaka et at.
1092
Materials and methods
Antibacterial agents
DU-6859a, levofloxacin, ciprofloxacin, and sparfloxacin were synthesized at the
New Product Research Laboratories I, Daiichi Pharmaceutical Co., Ltd., Tokyo,
Japan.
Organisms
Staphylococcus aureus FDA 209-P, methicillin-resistant S. aureus 900056 and 900477,
Streptococcus pneumoniae 57664, Escherichia coli K.L-16, and Pseudomonas aeruginosa
PAO1 were used.
Effect of growth conditions
The effects of variations in inoculum size and medium pH, as well as the addition of
metal ions and human serum, on DU-6859a activity were performed in Mueller-Hinton
agar (MHA; Difco Laboratories, Detroit, Mich.) by the dilution method (NCCLS,
1990). When testing S. pneumoniae, MHA supplemented 5% horse blood was used.
Calcium chloride, magnesium chloride, aluminum chloride, zinc chloride, manganese
chloride, ferrous chloride, and ferric chloride were added to Mueller-Hinton agar at
the final concentrations of 5 mM. One loopful (5 /iL) of an inoculum corresponding
to about 10*cfu per spot was inoculated on drug-containing agar plates, which
were then incubated for 18 h at 37°C. The MIC was defined as the lowest drug
concentration which prevented visible growth of bacteria. The effect of the medium was
determined by the agar dilution method with nutrient agar (NA; Eiken Co., Ltd.,
Tokyo, Japan), tryptone soy agar (TSA; Eiken), heart infusion agar (HIA; Eiken),
brain-heart infusion agar (BHIA; Difco) and Mueller-Hinton agar. The effect of human
1.56 -
A
\
~
0.39
\
—
\
\\
O
0.10 _-
n
-
\
\
0.026 -
\,
0.006 i
i
5.5
6
i
i
7
i
i
8
PH
Figure 1. Effects of medium pH on the activity of DU-6859a O . S- aureus FDA 209-P, Q, E. coli KL-16;
A- P- aeruginosa PAO1
1093
Antimicrobial activity of DU-6859a
Table. Effects of metal ions on the activity of 4-quinoloncs
MIC (mg/L)
CPFX
LVFX
Organism
Metal ion
DU-6859a
SPFX
5. aureus
FDA 209-P
none
calcium
magnesium
aluminum
zinc
manganese
ferrous
ferric
0.012
0.025
0.025
0.025
—
0.025
0.05
0.025
0.2
0.2
0.39
0.2
—
0.39
0.78
0.2
0.1
0.1
0.39
0.1
—
0.2
0.78
0.2
0.05
0.1
0.2
0.1
—
0.2
0.39
0.1
S. pneumoniae
57664
none
calcium
magnesium
aluminum
zinc
manganese
ferrous
ferric
0.1
0.1
0.2
0.1
—
0.2
0.2
0.1
1.56
1.56
3.13
1.56
—
1.56
3.13
1.56
3.13
6.25
12.5
6.25
—
6.25
12.5
3.13
0.39
0.39
0.78
0.39
—
0.78
0.78
0.39
E. coli
K.L-16
none
calcium
magnesium
aluminum
zinc
manganese
ferrous
ferric
0.025
0.025
0.05
0.025
—
0.05
0.1
0.025
0.05
0.1
0.2
0.1
—
0.2
0.39
0.05
0.025
0.05
0.1
0.025
—
0.1
0.39
0.05
0.025
0.05
0.05
0.05
—
0.1
0.2
0.05
P. aeruginosa
PAO1
none
calcium
magnesium
aluminum
zinc
manganese
ferrous
ferric
0.2
0.39
0.39
0.2
0.05
0.2
0.1
0.2
0.78
0.78
1.56
0.78
0.39
0.78
0.78
0.78
0.2
0.39
0.39
0.2
0.2
0.39
0.78
0.2
0.78
1.56
1.56
0.78
0.39
1.56
0.78
0.78
urine was examined by the macro dilution tube method (NCCLS, 1990) using
Mueller-Hinton broth (MHB; Difco) and 100% human urine (pH 5.9). Final inoculum
size was 5 x 10'cfu/mL.
Determination of bactericidal activity
The organisms incubated in Mueller-Hinton broth for 18 h at 37°C were diluted with
fresh broth to about 103 cfu/mL, and the diluted cultures were then incubated with
agitation for 2 h at 37°C. After this preincubation, cultures were incubated in the
absence of the indicated drug, or in its presence at levels equivalent to 1/4, 1/2, 1, 2,
and 4 times the MIC and, at time intervals of 0, 1, 2, 4, and 6 h, samples were removed,
serially diluted, and plated to determine the number of viable cells. The colonies were
counted after 24 h incubation at 37°C.
M. Tanaka et al.
1094
Samples of transmission electron microscopy (TEM) and scanning electron
microscopy (SEM) bacterial suspensions used in the above-mentioned experiment were
fixed with 1.5% glutaraldehyde and centrifuged at 6000 rpm for 20 min. The resulting
bacterial pellets were fixed with 1 % osmium tetroxide, and washed in the buffer of
Kellenberger, Ryter & Sechaud (1958), (pH 6.0). After that, for SEM, bacterial pellets
were dehydrated in graded ethanols, resuspended in isoamyl acetate, dried at the critical
point of carbon dioxide, and covered with platinum palladium. For TEM, bacterial
pellets were embedded in 2% purified agar, and small blocks were cut; these were stained
with 0.5% uranyl acetate buffer (pH 6.0) for 2 h, dehydrated for 30 min in graded
ethanols, embedded in epoxy resin (Luft, 1961), sectioned into slices about 70 nm thick,
and then stained again with uranyl acetate and lead citrate (Reynolds, 1963). Nuclear
staining was also performed with E. coli and observed by TEM (Zusman, Inouye &
Pardee, 1972).
Frequency of spontaneous resistance
The test strains were precultured in MHB for 20 h at 37°C until a concentration of
109cfu/mL was achieved. The bacterial suspension was plated onto MHA plates
containing two or four times the MIC of each compound. For testing S. pneumoniae,
MHB supplemented with 5% horse serum and MHA with 5% horse blood were used.
1 2
3
4
6
6
0
1 2
3
4
5
6
Time (h)
Flgnre 2. Bactericidal kinetics of DU-6859a. MICs were 0.012 mg/L for 5. aureus FDA 209-P (a);
0.05 mmg/L for methicillin-resistant S. aureus 900477 (b); 0.05 mg/L for 5. pneumoniae 57664 (c); 0.012 mg/L
for E. coli K.L-16 (d); and 0.20 mg/L for P. aeruginosa PAO1 (e). « . Control; O, 1/4 MIC; D, 1/2 MIC;
A- MIC; O. 2 x MIC; x , 4 x MIC.
Antimicrobial activity of DU-6859a
SEM
1095
TEM
(a)
(b)
(O
(d)
Figure 3. Electron micrographs of S. aureus FDA 209-P exposed to DU-6859a for 4 h. MIC was
0.012mg/L. (a) Control; (b) 1/4 MIC: (c) MIC; (d) 4 x MIC.
After 48 h at 37°C, colonies were counted and the frequency of spontaneous resistance
was calculated as the ratio of the number of colonies grown on drug-containing plates
to that on drug-free plates.
Development of resistance
Quinolones were prepared as two-fold dilutions in Mueller-Hinton broth. S. aureus
FDA 209-P, E. coli KL-16, and P. aeruginosa PAO1 were inoculated at 105 cfu/mL into
a dilution series of each drug. 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 procedure was repeated for ten days. The
resulting bacteria were tested for susceptibility to each of the drugs by the standard agar
dilution method described above.
M. Tanaka et al.
1096
Results
Effect of growth conditions on antibacterial activity
The MIC values of DU-6859a against S. aureus FDA 209-P, E. coli KL-16, and
P. aeruginosa PAO1 were closely similar in all of the five media, namely MuellerHinton, nutrient, heart infusion, brain-heart infusion, and tryptone soy agar (data not
shown). Alteration of the pH of Mueller-Hinton agar between 6 and 8, addition of
human serum at 10%, 25%, and 50% to the medium, and increase of the inoculum size
from 103 to 107 cfu each had no significant effect on the activity of DU-6859a against
any strain. However, activity of DU-6859a at pH 5.5 was between one-half and
one-sixteenth of the activity at higher pH, as were the activities of the other drugs tested
(Figure 1). In human urine, the MIC values against three strains of DU-6859a,
levofloxacin, ciprofloxacin, and sparfloxacin were 2-8, 4-16, 2-16, and 8-32 times
higher than the values in MHB, respectively. It was found that, irrespective of the
bacterial species tested, calcium chloride, aluminum chloride, manganese chloride, and
ferric chloride did not significantly alter the MIC of DU-6859a (Table). The MICs of
(a)
fb)
fc)
(d)
Figure 4. Transmission electron micrographs of 5. pneumoniae 57664 exposed to DU-6859a for 4 h. MIC
was 0.05 mg'L. (a) Control: (b) I 4 MIC: (c) MIC: (d) 4 x MIC.
Antimicrobial activity of DU-6859a
SEM
1097
TEM
(a)
(b)
(c)
(d)
Figure 5. Electron micrographs of E. coli KL-16 exposed to DU-6859a for 4 h. MIC was 0.012 mg/L. (a)
Control; (b) 1/4 MIC; (c) MIC; (d) 4 x MIC.
all compounds tested in the presence of magnesium chloride were two to four times
higher than the MICs without metal ions. Zinc chloride showed bactericidal activity
against the test strains. Against P. aeruginosa, metal ions did not antagonize any drug,
but ferrous salts antagonized all quinolones, and manganese chloride antagonized the
activity of the three reference drugs against E. coli.
Bactericidal activity
Killing curve studies showed that DU-6859a had rapid bactericidal activity against
S. aureus FDA 209-P, S. pneumoniae 57664, E. coli KL-16, and P. aeruginosa PAOI
at concentrations greater than the MIC (Figure 2). Levofloxacin, ciprofloxacin and
sparfloxacin showed similar bactericidal activity against these three strains. Against
methicillin-resistant S. aureus 900477, DU-6859a showed bactericidal activity at
M. Tanaka et al.
1098
concentrations greater than twice the MIC. Sparfloxacin showed only a 100-fold
decrease at the maximum at a concentration of 4 times the MIC. Examination of the
ultrastructure of untreated 5. aureus FDA 209-P cells in Figure 3 showed normal cell
walls and cytoplasm containing evenly dispersed ribosomes and nuclear material in
untreated cells. The typical alterations of S. aureus cells exposed to DU-6859a were
abnormal cell division and cell lysis. Normal S. pneumoniae cells had a thick capsule,
so that the cell surface could not be observed. Abnormal cell division and cell lysis were
observed as with S. aureus in treated cells (Figure 4). In the case of E. coli (Figure 5),
DU-6859a induced filamentation, cytoplasmic shrinkage and lysis. The cells of P.
aeruginosa PAO1 treated with DU-6859a appeared to be spheroplasts, blebs and lysed
cells (Figure 6). Nuclear staining showed the direct effect of DU-6859a on DNA
synthesis in E. coli (Figure 7). Small nuclei dispersed in filamentous cells were apparent
after exposure to the MIC of DU-6859a, and disappeared at 4 times the MIC.
SEM
TEM
(c)
(d)
Figure 6. Electron micrographs of P. aeruginosa PAOI exposed to DU-6859a for 4 h. MIC was 0.20 mg/L.
(a) Control: (b) 1 4 MIC; (c) MIC; (d) 4 x MIC.
Antimicrobial activity of DU-6859a
1099
(a)
(b)
(c)
(d)
Figure 7. Electron micrographs of E. coli KL-16 exposed to DU-6859a for 4 h. MIC was 0.012 mg/L.
(a) Control; (b) 1/4 MIC; (c) MIC; (d) 4 x MIC. Nuclear staining.
Frequency of spontaneous resistance
The frequency of spontaneous resistance of S. aureus FDA 209-P, S. pneumoniae
57664, E. coli KL-16, P. aeruginosa PAOl and the two strains of MRSA at twice the
MIC of DU-6859a were 4 . 4 x 1 0 - " , <5.0 x 10" 8 , < 1 . 9 x l O 9 , 7.1 x I O " \
2.7 x 10" 9 , and 4.2 x 10~\ respectively. Against sparfloxacin, a higher frequency of
resistance was shown by the two strains of MRSA (1.4 x 10" 6 and 9.1 x 10~6) than
to the other compounds. The frequency of S. pneumoniae resistance at twice the MIC
of sparfloxacin and ciprofloxacin was higher than that of DU-6859a and levofloxacin.
Except for the above cases, there were no particular differences in the frequency of
resistance of the species used to the quinolones tested.
Development of resistance
Against resistant strains selected after ten serial exposures to DU-6859a, this agent
showed MICs that were 4, 16, and 32 times those against the parent strain of S. aureus,
1100
M. Tanaka et al.
E. coli, and P. aeruginosa, respectively (Figure 8). The initial MIC values determined
by broth dilution were almost half of those determined by the agar dilution method.
Against resistant strains selected using levofloxacin, ciprofloxacin and sparfloxacin, on
the other hand, these agents showed MICs 4, 8, and 64 times those against the S. aureus
parent strain, respectively. For E. coli and P. aeruginosa, the corresponding values
ranged, respectively, from 16 to 64 times and from 32 to 128 times those against the
parent strains.
Discussion
In the past few years, a large number of fluoroquinolones have been developed (Hooper
& Wolfson, 1991). There has been a concerted effort to synthesize new agents with
improved activities, especially against Gram-positive bacteria. DU-6859a, a novel
quinolone antibacterial agent, has activity against a wide range of bacteria, and is
particularly effective against Gram-positive bacteria (Sato et al., 1992). The effects of
growth conditions on the antibacterial activity of DU-6859a were studied. The activity
of DU-6859a decreased at a low pH and in human urine, and there was a decrease in
activity in the presence of 5 mM ferrous ion. Smith reported the significant effect of 5 mM
aluminum, ferrous, and ferric ions on the activity of quinolones (Smith, 1990). In our
results, aluminum and ferric ions did not affect the activity of quinolones. It was
speculated that the difference of inoculum size was the main cause of the differences
in the results: Smith inoculated 20 to 100 cfu/plate, while we used 10*cfu/plate. The
influence of high concentrations (8.4 and 9.3 mM) of magnesium has been demonstrated
(Hirschhorn & Neu, 1986; Canton et al., 1992), and a similar result (decrease to between
one-fourth and one-eighth) was observed at 5 mM.
Time-kill curve studies showed the rapid bactericidal action of DU-6859a, but the
bactericidal activity of the drugs tested against methicillin-resistant S. aureus was the
weakest among the strains tested, especially with sparfloxacin. The bactericidal activity
of DU-6859a was similar to those of ciprofloxacin (Chalkley & Koornhof, 1985) and
AM-1155 (Hosaka et al., 1992). Morphological alterations of S. aureus, E. coli and
P. aeruginosa exposed to DU-6859a were similar to the alterations of cells treated with
ofloxacin, ciprofloxacin, and levofloxacin reported previously (Dougherty & Saukkonen,
1985; Elliott, Shelton & Greenwood, 1987; Tanaka, Otsuki & Nishino, 1992).
Figure 8. Development of resistance to DU-6859a. O, S. aureus FDA 209-P; • . E. coli K.L-16; A ,
aeruginosa PAO1.
Antimicrobial activity of DU-6859a
1101
In E. coli and P. aeruginosa, the frequency of spontaneous mutants resistant to
DU-6859a was similar to the frequency of resistance to levofloxacin, ciprofloxacin, and
sparfloxacin. However, the frequency of the resistance mutation of S. aureus to
DU-6859a was lower than those to ciprofloxacin and sparfloxacin. Furthermore, the
development of resistance after consecutive exposure of S. aureus to quinolones
demonstrated that the emergence of resistance to DU-6859a was slower than that to
sparfloxacin. These findings suggest the effectiveness of DU-6859a against various
bacterial infections.
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