Introduction

Topical application of antimicrobial agents is a useful tool
for the therapy of skin and soft-tissue infections. It has sev-
eral potential merits compared with systemic therapy.
Firstly, it can avoid an unnecessary exposure of the gut flora
(e.g. by the oral route), which may exert selection for resist-
ance. Secondly, it is expected that the high local drug con-
centration in topical application should overwhelm many
mutational resistances. Thirdly, topical applications are
less likely than systemic therapy to cause side effects.
At present, there are several kinds of antimicrobial
agent used in topical applications, such as β-lactams, quino-
lones, aminoglycosides, macrolides, tetracyclines, mupirocin
and fusidic acid. For chemotherapy of infection, antibiotic
resistance has been a major issue. In skin and soft-tissue
infections, the incidence of infections caused by multidrug-
resistant Gram-positive organisms, which are major patho-
gens in these infections, has been increasing, despite
advances in antimicrobial therapy over the last 20 years.
1
The incidence of staphylococci and streptococci resistant
to macrolides and aminoglycosides, in particular, has
increased markedly in recent years.
2,3
One of the reasons
for this is that resistance to these agents is easily spread
horizontally to other bacteria by R plasmids or trans-
posons. Recently, the emergence of plasmid-mediated
mupirocin resistance in methicillin-resistant Staphylococ-
cus aureus (MRSA) has also been reported.
4–6
Quinolones have a broad spectrum, display potent
antibacterial activity and are bactericidal against Gram-
positive and Gram-negative bacteria. Moreover, quinolones
show no cross-resistance to other classes of antimicrobials.
455
Original articles
In vitro and in vivo antibacterial activity of T-3912, a novel
non-fluorinated topical quinolone
Tetsumi Yamakawa*, Junichi Mitsuyama and Kazuya Hayashi
Research Laboratories of Toyama Chemical Co., 2-4-1 Shimookui, Toyama City, Toyama 930-8508, Japan
The in vitro and in vivo activity of T-3912, a novel non-fluorinated topical quinolone, was
compared with that of nadifloxacin, ofloxacin, levofloxacin, clindamycin, erythromycin and
gentamicin. The in vitro activity of T-3912 against methicillin-susceptible Staphylococcus
aureus, ofloxacin-resistant and methicillin-resistant S. aureus, Staphylococcus epidermidis,
ofloxacin-resistant S. epidermidis, penicillin-resistant Streptococcus pneumoniae and Pro-
pionibacterium acnes was four-fold to 16 000-fold greater than that of other agents at the
MIC
90
for the clinical isolates. The activity of T-3912 was not influenced by grlA mutation in
S. aureus, and the degree of MIC increase of T-3912 for grlA–gyrA double and triple mutants
was lowest among the quinolones tested (nadifloxacin, levofloxacin and ofloxacin). The
inhibitory activity of T-3912 was compared with other quinolones for DNA gyrase and
topoisomerase IV of S. aureus SA113. T-3912 showed the greatest inhibitory activity for both
enzymes among the quinolones tested. The isolation frequency of spontaneous mutants
resistant to T-3912 was < 1.7 ؋ 10
؊9
and < 2.0 ؋ 10
؊9
for S. aureus SA113 and P. acnes JCM
6425, respectively. Furthermore, resistance to T-3912 could not be clearly detected in the
28th transfer by the serial passage method. T-3912 exhibited more potent bactericidal
activity against S. aureus and P. acnes than nadifloxacin and clindamycin in a short time
period. T-3912 in a 1% gel formulation showed good therapeutic activity against a burn
infection model caused by S. aureus SA113, P. acnes JCM6425 and multidrug-resistant
S. aureus F-2161. These results indicate that T-3912 is potentially a useful quinolone for the
treatment of skin and soft-tissue infections and that its potent bactericidal activity might be
able to shorten the treatment period.
*Corresponding author. Tel: ϩ81-76-431-8270; Fax: ϩ81-76-431-8208; E-mail: tetsumi_yamakawa@toyama-chemical.co.jp
© 2002 The British Society for Antimicrobial Chemotherapy
Journal of Antimicrobial Chemotherapy (2002) 49, 455–465
JAC
T. Yamakawa et al.
In recent years, the emergence of resistant bacteria has
proved problematic during and after quinolone therapy
for several types of infection. However, it has been found
that a new class of des-F(6)-quinolones/non-fluorinated
quinolones has good antibacterial activity.
7,8
In attempting
to select a new topical quinolone with a high level of
activity against Gram-positive organisms, including quino-
lone-resistant bacteria, T-3912 {1-cyclopropyl-8-methyl-7-
[5-methyl-6-(methylamino)-3-pyridinyl]-4-oxo-1,4-dihydro-
3-quinolinecarboxylic acid} (Figure 1) was chosen as a
potential topical quinolone candidate for development.
In this study, the in vitro and in vivo antibacterial activity
of T-3912 was compared with that of nadifloxacin, ofloxa-
cin, levofloxacin, clindamycin, erythromycin and genta-
micin against major skin pathogens.
Materials and methods
Antimicrobial agents
The following agents were used: T-3912, nadifloxacin,
ofloxacin, levofloxacin, clindamycin, erythromycin and
gentamicin. Ofloxacin (Sigma Chemical, St Louis, MO),
clindamycin (Sigma), erythromycin (Sigma) and genta-
micin (Schering-Plough, Osaka, Japan) were commercially
available. Nadifloxacin and levofloxacin were prepared in
our laboratories. The purity of these quinolones was above
99%, as measured by high-performance liquid chromato-
graphy.
For in vivo evaluation, the following were used: T-3912
1% gel, prepared in our laboratory, and commercially
available nadifloxacin 1% cream (Acuatim cream; Otsuka
Pharmaceutical, Tokyo, Japan), clindamycin 1% gel
(Cleocin T Topical Gel; Pharmacia & Upjohn, MI),
erythromycin 2% gel (A/T/S 2%; Hoechst-Roussel, NJ)
and gentamicin 0.1% cream (Gentacin cream, Schering-
Plough).
Organisms
All 223 clinical isolates used in this study were collected
from various hospitals and research institutes in Japan
from 1997 to 1998. They included 48 strains of S. aureus,
including 23 strains of ofloxacin-resistant MRSA; 53 strains
of Staphylococcus epidermidis, including 26 strains of
ofloxacin-resistant S. epidermidis; 42 strains of Streptococ-
cus pneumoniae, including 22 strains of penicillin-resistant
S. pneumoniae; 26 strains of Streptococcus pyogenes;
27 strains of Pseudomonas aeruginosa; and 27 strains of
Propionibacterium acnes.
The quinolone-resistant S. aureus strains used in this
study were as follows: CR-3, obtained from a ciprofloxacin
first-step mutant of wild-type S. aureus SA113; and CRCP-
9, obtained from a ciprofloxacin second-step mutant of
strain CR-3 described previously.
9
F-1659, F-1614 and
F-2161 were obtained from clinical isolates from various
hospitals in Japan.
The nucleotide sequences of gyrA and grlA in the
quinolone resistance-determining region of these strains
were determined by dideoxy chain termination methods as
reported previously.
10
Animals
Three-and-a-half-week-old male ICR strain mice, pur-
chased from Japan SLC (Shizuoka, Japan), were assigned
to the study after an acclimatization period of 3 days.
Antibacterial activity
MICs were determined by the standard agar dilution
method or the broth microdilution method recommended
by the Japanese Society of Chemotherapy.
11
For the agar dilution method, Mueller–Hinton agar
(MHA; Difco, Detroit, MI) was employed for aerobic bac-
teria. The MHA was supplemented with 5% defibrinated
sheep blood (Nippon Bio-test Laboratories, Tokyo, Japan)
or 5% Fildes enrichment (Difco) to support the growth of
fastidious bacteria. Modified Gifu Anaerobe Medium
(GAM) agar (Nissui Seiyaku, Tokyo, Japan) was used for
anaerobic bacteria.
12
Almost all strains were tested at a
final inoculum of 10
4
cfu/spot by using a multipoint inocula-
tor (Sakuma Seisakusho, Tokyo, Japan). Aerobic bacteria
were incubated at 37ЊC for 18–24 h in air. Anaerobic bac-
teria were incubated in an anaerobic cabinet (Forma sci-
entific anaerobic system model) in an atmosphere of 10%
hydrogen, 10% carbon dioxide and 80% nitrogen. The
MIC was defined as the lowest antibiotic concentration that
prevented the visible growth of bacteria.
For the broth microdilution method, Mueller–Hinton
broth (MHB, Difco) cation-adjusted with calcium and
magnesium was used. Two-fold dilutions of antibiotics and
a final bacterial concentration of c. 5 ϫ10
4
cfu were placed
in each well and the plates incubated at 37ЊC overnight.
Again, the MIC was defined as the lowest concentration of
antibiotic that prevented visible growth.
Inhibition of DNA gyrase and topoisomerase IV
The genes encoding DNA topoisomerase IV (grlA and
grlB) and DNA gyrase (gyrAand gyrB) of S. aureus SA113
were cloned and expressed in Escherichia coli DH5␣, as a
fusion protein with maltose binding protein according to
the method described by Tanaka et al.
13
456
Figure 1. Chemical structure of T-3912.
In vitro and in vivo activity of T-3912
For inhibition of topoisomerase IV, the IC
50
was deter-
mined as the drug concentration that reduced decatenation
activity by 50%, as seen with drug-free controls. For inhibi-
tion of DNA gyrase, the IC
50
was determined as the drug
concentration that reduced supercoiling activity, i.e. the
conversion of relaxed pBR322 DNA to the supercoiled
form by 50%, as seen with the drug-free controls.
Determination of mutant frequency
The frequencies of occurrence of spontaneous mutants
resistant to T-3912, nadifloxacin, levofloxacin, clindamycin,
erythromycin and gentamicin in S. aureus SA113 and
P. acnes JCM6425 were determined by spreading a 0.1 mL
sample of a culture of each test organism on to MHA plates
for S. aureus and modified GAM agar plates for P. acnes
containing drugs at concentrations of 4 ϫ MIC. After
incubation at 37ЊC for 48 h, the colonies were counted
and the frequency of occurrence of spontaneously resistant
mutants was calculated as the ratio of the number of resist-
ant cells to the number of cells inoculated.
14
In vitro development of resistance
In vitro development of resistance was carried out using a
broth microdilution method by exposing bacteria to step-
wise increasing concentrations of antibiotics by slight
modification of the method of Entenza et al.
15
Selection
of drug-resistant derivatives was carried out by exposure of
S. aureus SA113 and P. acnes JCM6425 to stepwise increas-
ing concentrations of antibiotic. A series of microtitre wells
containing two-fold serial dilutions of each test drug was
inoculated with a final concentration of 10
5
cfu/mL and
then incubated for 24 h for S. aureus SA113 and for 48 h
for P. acnes JCM6425. In the next step, the well with the
highest antibiotic concentration still showing turbidity was
used to inoculate a new series of microtitre tray. The pro-
cedure was repeated, and the MICs were determined for up
to 28 passages.
Bactericidal activity
In order to assess bactericidal activity, the log
10
reduction
of bacterial counts in a definite time was investigated with a
range of drug concentrations of 1–32 ϫ MIC, to give an
inoculum size of c. 10
6
cfu/mL at 37ЊC. The incubation
period was 2 h for S. aureus SA113 and 4 h for P. acnes
JCM6425. After incubation, viable counts were made on
solid agar. Experiments were carried out in triplicate, and
the log
10
reduction of viable counts was given as the mean
Ϯstandard deviation.
In vivo therapeutic efficacy
This animal study was approved by the Internal Ethics
Committee of Toyama Chemical on Animal Studies and
carried out according to the procedures stipulated by it.
The therapeutic effect of T-3912 was evaluated by the burn
infection model according to Kawabata et al.
16
Two drug-
susceptible strains, S. aureus SA113 and P. acnes JCM6425,
and one multidrug-resistant strain, S. aureus F-2161, were
used. Briefly, using a sterilized cotton swab, bacterial cells
grown on MHA plates were suspended in sterilized physio-
logical saline at the desired concentration (confirmed by
placing serial 10-fold dilutions on to MHA and incubating
the resulting plates for 18 h at 37ЊC). The four-week-old
male ICR strain mice were anaesthetized by intramuscular
injection of a mixture of 6 mg of ketamine (Sankyo Pharma-
ceutical, Tokyo, Japan) per kilogram of body weight and
1 mg of xylazine (Bayer, Tokyo, Japan) per kilogram.
Their dorsal hair was removed by an electric shaver. A
metal weight (20 mm in diameter) heated to 100ЊC was
pressed on the dorsal skin for 5 s. After 1 h, 0.2 mL of
bacterial suspension was subcutaneously injected at the
burned skin site. At 2 h after infection, 10 mg of antimicro-
bial formulation (T-3912 1% gel, nadifloxacin 1% cream,
clindamycin 1% gel, erythromycin 2% gel or gentamicin
0.1% cream) was applied to the lesion. Mice were
humanely killed 24 h after this for S. aureus SA113 and
P. acnes JCM6425 and 48 h after this for S. aureus F-2161.
After cleaning the surface of the burned skin lesion using
70% alcohol, the burned skin lesions were removed. They
were then homogenized with sterilized physiological saline
and the homogenate diluted with sterilized physiological
saline, placed on to MHA plates containing 50 mM MgCl
2
to avoid carrying over the quinolones. The number of
colonies was counted after incubation for 24 h at 37ЊC. The
results were expressed as the mean Ϯstandard deviation of
log cfu per skin sample. The lower limit of detection was
10
4
cfu/skin sample.
Statistical analysis
The log
10
reduction of viable cell counts was analysed by
the Tukey procedure with a cutoff of P ϭ 0.05 for signifi-
cance (SAS ver. 6.12; SAS Institute, Tokyo, Japan).
Results
Antibacterial activity
Table 1 shows the MIC ranges and the MICs at which 50%
and 90% of the clinical isolates of Gram-positive and
Gram-negative bacteria were inhibited (MIC
50
and MIC
90
,
respectively). The antibacterial activity of T-3912 based on
MIC
90
values against methicillin-susceptible S. aureus,
ofloxacin-resistant MRSA, S. epidermidis, ofloxacin-resist-
ant S. epidermidis, penicillin-resistant S. pneumoniae
and P. acnes was between two- and Ͼ16 000-fold greater
than that of nadifloxacin, ofloxacin, levofloxacin, clinda-
mycin, erythromycin and gentamicin. The activity of
T-3912 against penicillin-susceptible S. pneumoniae
457
T. Yamakawa et al.
458
Table 1. Antibacterial activity of T-3912 and other agents against clinical isolates
Organism Antibacterial Range of MIC MIC
50
MIC
90
(number of strains) agent (mg/L) (mg/L) (mg/L)
S. aureus T-3912 0.00313–0.00625 0.00625 0.00625
methicillin-susceptible (25) nadifloxacin 0.0125–0.05 0.025 0.05
ofloxacin 0.2–1.56 0.39 0.78
levofloxacin 0.1–0.39 0.2 0.39
clindamycin 0.1–>100 0.1 0.2
erythromycin 0.2–>100 0.2 >100
gentamicin 0.05–100 0.39 100
S. aureus T-3912 0.025–0.2 0.2 0.2
methicillin-resistant, nadifloxacin 0.78–1.56 1.56 1.56
ofloxacin-resistant (23) ofloxacin 12.5–100 50 50
levofloxacin 3.13–50 25 25
clindamycin >100 >100 >100
erythromycin >100 >100 >100
gentamicin 0.2–>100 25 100
S. epidermidis T-3912 0.00625–0.025 0.00625 0.0125
(27) nadifloxacin 0.025–0.05 0.05 0.05
ofloxacin 0.20–0.78 0.39 0.39
levofloxacin 0.10–0.39 0.20 0.20
clindamycin 0.05–>100 0.1 1.56
erythromycin 0.025–>100 0.1 >100
gentamicin 0.025–100 0.1 50
S. epidermidis T-3912 0.05–0.2 0.1 0.2
ofloxacin-resistant nadifloxacin 0.78–1.56 0.78 1.56
(26) ofloxacin 6.25–50 6.25 25
levofloxacin 3.13–25 3.13 12.5
clindamycin 0.025–>100 0.1 >100
erythromycin 0.05–>100 50 >100
gentamicin 0.05–>100 50 100
S. pneumoniae T-3912 0.0125–0.1 0.05 0.1
penicillin-susceptible (20) nadifloxacin 0.39–3.13 0.78 1.56
ofloxacin 1.56–6.25 1.56 3.13
levofloxacin 0.78–3.13 0.78 1.56
clindamycin 0.00313–0.05 0.025 0.025
erythromycin 0.00313–3.13 0.025 0.39
gentamicin 3.13–12.5 6.25 12.5
S. pneumoniae T-3912 0.025–0.05 0.05 0.05
penicillin-resistant (22) nadifloxacin 0.78–1.56 0.78 1.56
ofloxacin 0.78–1.56 0.78 1.56
levofloxacin 0.39–0.78 0.39 0.39
clindamycin 0.025–>100 0.025 >100
erythromycin 0.0125–>100 0.05 >100
gentamicin 6.25–12.5 12.5 12.5
S. pyogenes T-3912 0.00625–0.05 0.025 0.05
(26) nadifloxacin 0.1–1.78 0.39 0.78
ofloxacin 0.1–3.13 0.78 3.13
levofloxacin 0.2–1.56 0.39 1.56
clindamycin 0.0125–0.1 0.05 0.1
erythromycin 0.00625–0.1 0.025 0.05
gentamicin 0.2–12.5 0.78 12.5
In vitro and in vivo activity of T-3912
was inferior to that of clindamycin and four- to 128-fold
greater than that of nadifloxacin, ofloxacin, levofloxacin,
erythromycin and gentamicin. The activity of T-3912
against S. pyogenes was comparable to that of erythromycin
and two- to 256-fold greater than that of nadifloxacin,
ofloxacin, levofloxacin, clindamycin and gentamicin. The
activity of T-3912 against P. aeruginosa was inferior to
that of ofloxacin and levofloxacin but superior to that of
nadifloxacin, clindamycin, erythromycin and gentamicin.
Mechanisms of action
Table 2 shows the antibacterial activity of T-3912 and
reference quinolones against S. aureus having mutations at
the grlA and gyrA locus. The MICs of ofloxacin and levo-
floxacin increased four-fold for the grlA mutant, 16-
to 128-fold for the grlA–gyrA double mutants and 512- to
у1024-fold for the grlA–gyrA triple mutant.
The MIC of nadifloxacin for the grlA mutant did not
alter from that for the wild-type strain but increased eight-
to 64-fold for the grlA–gyrAdouble mutants and 1024-fold
for the grlA–gyrA triple mutant. In a similar manner, the
MIC of T-3912 for the grlA mutant was not different from
that for the wild-type strain but increased two- to eight-fold
for the grlA–gyrA double mutants and 32-fold for the
grlA–gyrA triple mutant. The activity of T-3912 was not
influenced by the grlA mutation, and the degree of
MIC increase for the grlA–gyrA double mutants and the
grlA–gyrA triple mutant in T-3912 was the lowest among
the quinolones tested.
Table 3 shows the MICs and the inhibitory activity of
T-3912 and other reference quinolones for DNA gyrase
and topoisomerase IV obtained from S. aureus SA113. The
IC
50
values of T-3912 for DNA gyrase and topoisomerase
IV were 4.50 and 0.617 mg/L, respectively. For both DNA
gyrase and topoisomerase IV, T-3912 showed the greatest
inhibitory activity among the quinolones tested.
Development of resistance
Table 4 shows the isolation frequency of spontaneous
mutants in S. aureus SA113 and P. acnes JCM6425 resistant
to T-3912 and other agents.
In S. aureus, the isolation frequency of mutants resistant
to T-3912 was Ͻ1.7 ϫ 10
Ϫ9
, lower than that of gentamicin
and levofloxacin and comparable to that of nadifloxacin,
clindamycin and erythromycin. In P. acnes, the isolation
frequency of mutants resistant to T-3912 was Ͻ2.0 ϫ 10
Ϫ9
,
comparable to that of other agents. In general, the isolation
rate of mutants resistant to T-3912 was quite low, similar to
that of nadifloxacin, clindamycin and erythromycin.
Figure 2 shows the in vitro development of resistance to
T-3912, nadifloxacin, levofloxacin, clindamycin, erythro-
mycin and gentamicin in S. aureus SA113 and P. acnes
JCM6425 by the serial passage method. The increase of
MICs through 28 passages in S. aureus and P. acnes was
рtwo-fold for T-3912, рfour-fold for nadifloxacin, four- to
64-fold for levofloxacin, two- to four-fold for clindamycin,
16-fold for erythromycin and four- to eight-fold for genta-
micin.
Bactericidal activity
Figure 3 shows the log
10
reduction in bacterial counts of
S. aureus SA113 and P. acnes JCM6425 when exposed to
each agent at concentrations of 1–32 ϫMIC.
459
Table 1. (Continued)
Organism Antibacterial Range of MIC MIC
50
MIC
90
(number of strains) agent (mg/L) (mg/L) (mg/L)
P. aeruginosa T-3912 0.1–50 1.56 6.25
(27) nadifloxacin 0.2–>100 3.13 12.5
ofloxacin 0.2–>100 0.78 3.13
levofloxacin 0.1–>100 0.39 3.13
clindamycin >100 >100 >100
erythromycin 25–>100 >100 >100
gentamicin 0.39–>100 1.56 12.5
P. acnes T-3912 0.00625–0.05 0.025 0.05
(27) nadifloxacin 0.2–0.39 0.39 0.39
ofloxacin 0.78–1.56 0.78 1.56
levofloxacin 0.39–0.78 0.39 0.78
clindamycin 0.05–0.78 0.1 0.78
erythromycin 0.0125–0.78 0.05 0.1
gentamicin 1.56–12.5 6.25 12.5
Inoculum size: 10
4
cfu/spot.
T. Yamakawa et al.
Against S. aureus SA113, the bactericidal activity of
T-3912 was inferior to that of gentamicin but superior to
that of nadifloxacin and clindamycin at concentrations
of 4–32 and 1–32 ϫMIC, respectively.
Against P. acnes JCM6425, although the activity of
T-3912 was again inferior to that of gentamicin at a con-
centration of 32 ϫ MIC, it was superior to that of nadi-
floxacin and clindamycin at concentrations of 4–32 and
460
Table 2. Antibacterial activity of T-3912 and other quinolones against quinolone-resistant S. aureus
Mutations at QRDRs
a
MIC (mg/L)
b
Strain GyrA GrlA T-3912 nadifloxacin ofloxacin levofloxacin
SA113 (wild-type) 0.0078 0.0313 0.25 0.125
CR-3 Ser
83
ǞPhe 0.0078 0.0313 1 0.5
F-1659 Glu
88
ǞGly Ser
80
ǞTyr 0.0156 0.25 4 2
CRCP-9 Glu
88
ǞLys Ser
80
ǞPhe 0.0625 0.5 8 4
F-1614 Ser
84
ǞLeu Glu
84
ǞLys 0.0625 2 32 16
F-2161 Ser
84
ǞLeu, Glu
88
ǞLys Ser
80
ǞPhe 0.25 32 >128 >128
a
Quinolone resistance-determining regions.
b
10
4
cfu/well.
Table 3. Inhibitory activity of T-3912, nadifloxacin, ofloxacin and levofloxacin
against DNA gyrase and topoisomerase IV obtained from S. aureus SA113
IC
50
(mg/L)
Drugs MIC (mg/L)
a
DNA gyrase topoisomerase IV
T-3912 0.0078 4.50 0.617
Nadifloxacin 0.0313 8.83 3.22
Ofloxacin 0.25 119 3.59
Levofloxacin 0.125 39.7 1.70
a
10
4
cfu/well.
Table 4. Frequency of spontaneous mutants resistant to T-3912 and other agents
Organism Drug MIC (mg/L)
a
Frequency (ϫ10
Ϫ9
)
(4 ϫMIC)
S. aureus SA113 T-3912 0.00625 <1.7
nadifloxacin 0.025 <1.7
levofloxacin 0.1 420
clindamycin 0.1 <1.7
erythromycin 0.2 <1.7
gentamicin 0.2 1800
P. acnes JCM6425 T-3912 0.05 <2.0
nadifloxacin 0.39 <2.0
levofloxacin 0.78 <2.0
clindamycin 0.1 <2.0
erythromycin 0.05 <2.0
gentamicin 3.13 <2.0
a
10
4
cfu/spot.
In vitro and in vivo activity of T-3912
1–32 ϫ MIC, respectively. In most cases, gentamicin
decreased the viable counts most potently against these
strains, followed by T-3912 and nadifloxacin. Clindamycin
showed bacteriostatic activity against these strains. T-3912
exhibited more potent bactericidal activity than nadi-
floxacin and clindamycin against S. aureus and P. acnes.
Efficacy for experimental burn infection
Figure 4 shows the efficacy of T-3912 1% gel formulation
and each commercially available formulation of the other
agents on an experimental burn infection model caused by
S. aureus SA113 and P. acnes JCM6425. For the infection
caused by S. aureus SA113, T-3912 1% gel, nadifloxacin 1%
cream, clindamycin 1% gel and erythromycin 2% gel all
significantly decreased the bacterial count in the skin lesion
compared with the control (P Ͻ0.01). Clindamycin 1% gel
significantly decreased bacterial counts compared with
nadifloxacin 1% cream (P Ͻ 0.05), erythromycin 2% gel
(P Ͻ0.05) and gentamicin 0.1% gel (P Ͻ0.01). T-3912 1%
gel also significantly decreased bacterial counts compared
with gentamicin 0.1% gel (P Ͻ 0.01). For the infection
caused by P. acnes JCM6425, once again all drugs tested
significantly decreased bacterial counts compared with the
control (P Ͻ 0.01), but in this case T-3912 1% gel showed
superior efficacy in reducing the bacterial count compared
with nadifloxacin 1% cream, clindamycin 1% gel, erythro-
mycin 2% gel and gentamicin 0.1% gel (P Ͻ0.01).
Figure 5 shows the case of infection caused by the
grlA–gyrA triple mutant S. aureus F-2161. As shown in
the Figure, only T-3912 1% gel decreased log cfu/skin at
the burned lesion significantly compared with not only the
control but also the other formulations tested.
Discussion
This study demonstrated the in vitro and in vivo activity of
T-3912, a novel topical non-fluorinated quinolone. Most
skin and soft-tissue infections are caused by Gram-positive
organisms, i.e. S. aureus and the β-haemolytic Streptococcus
species. MRSA, in particular, remains a serious cause of
infection. Therefore, we evaluated the antibacterial activ-
ity of T-3912 against Gram-positive organisms, including
drug-resistant bacteria.
461
Figure 2. Development of resistance in (a) S. aureus SA113 and (b) P. acnes JCM6425 against T-3912 and other agents by serial
passage method: ᭹, T-3912; ᭝, nadifloxacin; ᭡, levofloxacin; ᭿, clindamycin; ٗ, erythromycin; ϫ, gentamicin.
T. Yamakawa et al.
T-3912 showed the greatest antibacterial activity against
clinical isolates of Gram-positive organisms, i.e. methicillin-
susceptible S. aureus, ofloxacin-resistant MRSA, S. epider-
midis, ofloxacin-resistant S. epidermidis, penicillin-resistant
S. pneumoniae and P. acnes. In addition, T-3912 showed
improved activity against S. aureus with mutations in the
DNA gyrase and topoisomerase IV.
The target molecules of quinolones are DNA gyrase
and topoisomerase IV. The development of quinolone
resistance is caused by point mutations in discrete regions
of the DNA gyrase and topoisomerase IV genes called the
quinolone resistance-determining regions.
17,18
Quinolone
resistance in S. aureus arises through mutation of the parC
(grlA) or parE (grlB) genes before changes in the DNA
gyrase genes take place, indicating that topoisomerase IV
is the primary target and that DNA gyrase is the secondary
target in this Gram-positive bacteria.
19–25
However, in the case of T-3912, there are some interest-
ing characteristics. Firstly, the MIC of T-3912 did not alter
for the grlA mutant compared with the wild type, and the
degree of MIC increase for the grlA–gyrA double mutant
and the grlA–gyrA triple mutant was lowest among the
462
Figure 3. Bactericidal activity of T-3912, nadifloxacin, clindamycin and gentamicin against (a) S. aureus SA113 and (b) P. acnes
JCM6425. Results expressed as the mean Ϯ S.D. (n ϭ 3); P Ͻ 0.05. (a) Dotted line represents the control after incubation for 2 h in
S. aureus SA113; ᭹, T-3912 (MIC 0.0078 mg/L); ᭝, nadifloxacin (MIC 0.0625 mg/L); ᭿, clindamycin (MIC 0.125 mg/L); ϫ, gentamicin
(MIC 0.5 mg/L); T-3912 versus nadifloxacin (4–32 ϫ MIC), T-3912 versus clindamycin (1–32 ϫ MIC), T-3912 versus gentamicin
(1–32 ϫ MIC), nadifloxacin versus clindamycin (1–32 ϫ MIC), nadifloxacin versus gentamicin (1–32 ϫ MIC), clindamycin versus
gentamicin (1–32 ϫMIC). (b) Dotted line represents the control after incubation for 4 h in P. acnes JCM6425; ᭹, T-3912 (MIC
0.0313 mg/L); ᭝, nadifloxacin (MIC 1 mg/L); ᭿, clindamycin (MIC 0.0625 mg/L); ϫ, gentamicin (MIC 4 mg/L); T-3912 versus
nadifloxacin (4–32 ϫ MIC), T-3912 versus clindamycin (2–32 ϫ MIC), T-3912 versus gentamicin (32 ϫ MIC), nadifloxacin versus
gentamicin (8–32 ϫMIC), clindamycin versus gentamicin (4–32 ϫMIC).
In vitro and in vivo activity of T-3912
quinolones tested. Secondly, the IC
50
of T-3912 for both
topoisomerase IV and DNA gyrase was lowest among the
quinolones tested, reflecting the MIC. Thirdly, the isolation
frequency of spontaneous mutants resistant to T-3912 was
quite low, and resistance to T-3912 was not clearly detected
using the serial passage method. These results indicate that
the target of T-3912 may be both DNA gyrase and topoiso-
merase IV in S. aureus. Although further investigation is
needed on this matter, from the viewpoint of bacterial
resistance, this characteristic is very advantageous for the
treatment of infectious diseases, including skin and soft-
tissue infections.
In skin and soft-tissue infections, it is also necessary to
reduce the bacterial counts as rapidly as possible from the
infectious lesion. Therefore, rapid bactericidal activity is
one of the important characteristics of topical agents
required for an optimal in vivo therapeutic effect. T-3912
showed greater bactericidal activity than nadifloxacin and
clindamycin against S. aureus and P. acnes, which are the
typical pathogens of skin and soft-tissue infections and
acne, in a short time period. In addition, T-3912 1% gel
showed a superior therapeutic effect in the burn infection
model compared with gentamicin 0.1% cream for S. aureus
and with nadifloxacin 1% cream, clindamycin 1% gel, ery-
thromycin 2% gel and gentamicin 0.1% gel for P. acnes.
Furthermore, there is a report that the incidence of MRSA
has increased, with strains shown to cause up to 21% of skin
infection.
26
Therefore, efficacy against MRSA will become
more important for the treatment of skin infections. In
the burn infection model of S. aureus F-2161, a highly
multidrug-resistant strain including methicillin resistance
with grlA and gyrA triple mutation, only T-3912 1% gel
showed a therapeutic effect, which was reflected in its
MIC.
On the other hand, it is reasonable to consider that the
important factors determining the in vivo efficacy are not
only antibacterial activity and bactericidal activity but also
the tissue distribution of these drugs depending on formu-
lation. Indeed, the bactericidal activity of gentamicin was
more potent than that of T-3912, but the therapeutic effect
of gentamicin was inferior to that of T-3912. This result
indicated that the tissue concentration of gentamicin in the
infectious lesion was lower than that of T-3912. Moreover,
the permeability of gentamicin through the stratum
corneum might be poorer than T-3912 due to its formula-
tion. Hence, there is a need to investigate in detail the rela-
tionship between the pharmacokinetic parameters of these
topical agents and their formulations in skin.
In conclusion, T-3912 is a potentially useful quinolone
for the treatment of skin and soft-tissue infections, and its
potent bactericidal activity might be able to shorten the
treatment period of such infections.
Acknowledgements
The authors would like to thank H. Yamada, R. Kitayama,
Y. Furuta, Y. Yamashiro, M. Yonezawa, M. Nakata, H.
Yamada, H. Hisada, Y. Shinmura, N. Annen and J. Mae-
hana for their technical assistance. We thank H. Kuroda
and H. Kawabuchi for the supply of chemical compounds.
We are also grateful to S. Kato, M. Katai and M. Kadono
for the preparation of formulations.
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Figure 4. Effect of T-3912 on lesions subcutaneously infected
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465

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