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


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 Propionibacterium acnes was four-fold to 16 000-fold greater than that of other agents at the MIC90 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 grlAgyrA 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 x 10-9 and < 2.0 x 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.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Topical application of antimicrobial agents is a useful tool for the therapy of skin and soft-tissue infections. It has several 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 resistance. Secondly, it is expected that the high local drug concentration 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 ß-lactams, quinolones, 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 pathogens 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 transposons. Recently, the emergence of plasmid-mediated mupirocin resistance in methicillin-resistant Staphylococcus aureus (MRSA) has also been reported.4–6

Quinolones have a broad spectrum, display potent antibacterial activity and are bactericidal against Grampositive and Gram-negative bacteria. Moreover, quinolones show no cross-resistance to other classes of antimicrobials. 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 quinolone-resistant bacteria, T-3912 {1-cyclopropyl-8-methyl-7- [5-methyl-6-(methylamino)-3-pyridinyl]-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid} (Figure 1Go) was chosen as a potential topical quinolone candidate for development.



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Figure 1. Chemical structure of T-3912.

 
In this study, the in vitro and in vivo antibacterial activity of T-3912 was compared with that of nadifloxacin, ofloxacin, levofloxacin, clindamycin, erythromycin and gentamicin against major skin pathogens.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 gentamicin (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 chromatography.

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 Streptococcus 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, purchased 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 bacteria. 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 104 cfu/spot by using a multipoint inoculator (Sakuma Seisakusho, Tokyo, Japan). Aerobic bacteria were incubated at 37°C for 18–24 h in air. Anaerobic bacteria were incubated in an anaerobic cabinet (Forma scientific 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 x 104 cfu were placed in each well and the plates incubated at 37°C 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 (gyrA and gyrB) of S. aureus SA113 were cloned and expressed in Escherichia coli DH5{alpha}, as a fusion protein with maltose binding protein according to the method described by Tanaka et al.13

For inhibition of topoisomerase IV, the IC50 was determined as the drug concentration that reduced decatenation activity by 50%, as seen with drug-free controls. For inhibition of DNA gyrase, the IC50 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 x MIC. After incubation at 37°C 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 resistant 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 stepwise 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 increasing concentrations of antibiotic. A series of microtitre wells containing two-fold serial dilutions of each test drug was inoculated with a final concentration of 105 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 procedure was repeated, and the MICs were determined for up to 28 passages.

Bactericidal activity

In order to assess bactericidal activity, the log10 reduction of bacterial counts in a definite time was investigated with a range of drug concentrations of 1–32 x MIC, to give an inoculum size of c. 106 cfu/mL at 37°C. 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 log10 reduction of viable counts was given as the mean ± standard 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 physiological saline at the desired concentration (confirmed by placing serial 10-fold dilutions on to MHA and incubating the resulting plates for 18 h at 37°C). The four-week-old male ICR strain mice were anaesthetized by intramuscular injection of a mixture of 6 mg of ketamine (Sankyo Pharmaceutical, 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 100°C 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 antimicrobial 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 MgCl2 to avoid carrying over the quinolones. The number of colonies was counted after incubation for 24 h at 37°C. The results were expressed as the mean ± standard deviation of log cfu per skin sample. The lower limit of detection was 104 cfu/skin sample.

Statistical analysis

The log10 reduction of viable cell counts was analysed by the Tukey procedure with a cutoff of P = 0.05 for significance (SAS ver. 6.12; SAS Institute, Tokyo, Japan).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Antibacterial activity

Table 1Go 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 (MIC50 and MIC90, respectively). The antibacterial activity of T-3912 based on MIC90 values against methicillin-susceptible S. aureus, ofloxacin-resistant MRSA, S. epidermidis, ofloxacin-resistant S. epidermidis, penicillin-resistant S. pneumoniae and P. acnes was between two- and >16 000-fold greater than that of nadifloxacin, ofloxacin, levofloxacin, clindamycin, erythromycin and gentamicin. The activity of T-3912 against penicillin-susceptible S. pneumoniae 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.


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Table 1. Antibacterial activity of T-3912 and other agents against clinical isolates
 
Mechanisms of action

Table 2Go 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 levofloxacin increased four-fold for the grlA mutant, 16- to 128-fold for the grlAgyrA double mutants and 512- to >=1024-fold for the grlAgyrA triple mutant.


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Table 2. Antibacterial activity of T-3912 and other quinolones against quinolone-resistant S. aureus
 
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 grlAgyrA double mutants and 1024-fold for the grlAgyrA 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 grlAgyrA double mutants and 32-fold for the grlAgyrA triple mutant. The activity of T-3912 was not influenced by the grlA mutation, and the degree of MIC increase for the grlAgyrA double mutants and the grlAgyrA triple mutant in T-3912 was the lowest among the quinolones tested.

Table 3Go 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 IC50 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.


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Table 3. Inhibitory activity of T-3912, nadifloxacin, ofloxacin and levofloxacin against DNA gyrase and topoisomerase IV obtained from S. aureus SA113
 
Development of resistance

Table 4Go shows the isolation frequency of spontaneous mutants in S. aureus SA113 and P. acnes JCM6425 resistant to T-3912 and other agents.


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Table 4. Frequency of spontaneous mutants resistant to T-3912 and other agents
 
In S. aureus, the isolation frequency of mutants resistant to T-3912 was <1.7 x 10-9, 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 <2.0 x 10-9, 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 2Go shows the in vitro development of resistance to T-3912, nadifloxacin, levofloxacin, clindamycin, erythromycin 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 <=two-fold for T-3912, <=four-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 gentamicin.



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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; {triangleup}, nadifloxacin; {blacktriangleup}, levofloxacin; {blacksquare}, clindamycin; {square}, erythromycin; x, gentamicin.

 
Bactericidal activity

Figure 3Go shows the log10 reduction in bacterial counts of S. aureus SA113 and P. acnes JCM6425 when exposed to each agent at concentrations of 1–32 x MIC.



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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 ± s.d. (n = 3); P < 0.05. (a) Dotted line represents the control after incubation for 2 h in S. aureus SA113; •, T-3912 (MIC 0.0078 mg/L); {triangleup}, nadifloxacin (MIC 0.0625 mg/L); {blacksquare}, clindamycin (MIC 0.125 mg/L); x, gentamicin (MIC 0.5 mg/L); T-3912 versus nadifloxacin (4–32 x MIC), T-3912 versus clindamycin (1–32 x MIC), T-3912 versus gentamicin (1–32 x MIC), nadifloxacin versus clindamycin (1–32 x MIC), nadifloxacin versus gentamicin (1–32 x MIC), clindamycin versus gentamicin (1–32 xMIC). (b) Dotted line represents the control after incubation for 4 h in P. acnes JCM6425; •, T-3912 (MIC 0.0313 mg/L); {triangleup}, nadifloxacin (MIC 1 mg/L); {blacksquare}, clindamycin (MIC 0.0625 mg/L); x, gentamicin (MIC 4 mg/L); T-3912 versus nadifloxacin (4–32 x MIC), T-3912 versus clindamycin (2–32 x MIC), T-3912 versus gentamicin (32 x MIC), nadifloxacin versus gentamicin (8–32 x MIC), clindamycin versus gentamicin (4–32 xMIC).

 
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 x MIC, respectively.

Against P. acnes JCM6425, although the activity of T-3912 was again inferior to that of gentamicin at a concentration of 32 x MIC, it was superior to that of nadifloxacin and clindamycin at concentrations of 4–32 and 1–32 x 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 nadifloxacin and clindamycin against S. aureus and P. acnes.

Efficacy for experimental burn infection

Figure 4Go 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 < 0.01). Clindamycin 1% gel significantly decreased bacterial counts compared with nadifloxacin 1% cream (P < 0.05), erythromycin 2% gel (P < 0.05) and gentamicin 0.1% gel (P < 0.01). T-3912 1% gel also significantly decreased bacterial counts compared with gentamicin 0.1% gel (P < 0.01). For the infection caused by P. acnes JCM6425, once again all drugs tested significantly decreased bacterial counts compared with the control (P < 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, erythromycin 2% gel and gentamicin 0.1% gel (P < 0.01).



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Figure 4. Effect of T-3912 on lesions subcutaneously infected with (a) S. aureus SA113 and (b) P. acnes JCM6425 in mice. Mice were infected subcutaneously with 0.2 mL of bacterial suspension (S. aureus SA113, 1.0 x 107 cfu/mouse; P. acnes JCM6425, 5.0 x 106 cfu/mouse). Drugs were applied at 2 h after infection (10 mg gel or cream per mouse). Viable cells were counted at 24 h after infection. The MICs for S. aureus SA113 were as follows: T-3912, 0.0078 mg/L; nadifloxacin, 0.0625 mg/L; clindamycin, 0.125 mg/L; erythromycin, 0.25 mg/L; gentamicin, 0.5 mg/L. The MICs for P. acnes JCM6425 were as follows: T-3912, 0.0313 mg/L; nadifloxacin, 0.25 mg/L; clindamycin, 0.0625 mg/L; erythromycin, 0.0625 mg/L; gentamicin, 2 mg/L. Statistical analysis was carried out using the Tukey procedure.

 
Figure 5Go shows the case of infection caused by the grlAgyrA 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.



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Figure 5. Effect of T-3912 on lesions subcutaneously infected with quinolone-, clindamycin-, erythromycin- and gentamicin-resistant S. aureus F-2161 in mice. Mice were infected subcutaneously with 0.2 mL of bacterial suspension (4.4 x 107 cfu/mouse). Drugs were applied at 2 h after infection (10 mg gel or cream per mouse). The MICs for S. aureus F-2161 were as follows: T-3912, 0.25 mg/L; nadifloxacin, 32 mg/L; clindamycin, >128 mg/L; erythromycin, 64 mg/L; gentamicin, 64 mg/L. Statistical analysis was carried out using the Tukey procedure.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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 ß-haemolytic Streptococcus species. MRSA, in particular, remains a serious cause of infection. Therefore, we evaluated the antibacterial activity of T-3912 against Gram-positive organisms, including drug-resistant bacteria.

T-3912 showed the greatest antibacterial activity against clinical isolates of Gram-positive organisms, i.e. methicillin-susceptible S. aureus, ofloxacin-resistant MRSA, S. epidermidis, 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 interesting 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 grlAgyrA double mutant and the grlAgyrA triple mutant was lowest among the quinolones tested. Secondly, the IC50 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 topoisomerase 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 softtissue 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, erythromycin 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 formulation. 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 formulation. Hence, there is a need to investigate in detail the relationship 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
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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. Maehana 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.


    Notes
 
* Corresponding author. Tel: +81-76-431-8270; Fax: +81-76-431-8208; E-mail: tetsumi_yamakawa{at}toyama-chemical.co.jp Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
1 . Baquero, F. (1997). Gram-positive resistance: challenge for the development of new antibiotics. Journal of Antimicrobial Chemotherapy39, Suppl. A, 1–6.[Free Full Text]

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3 . Schmitz, F. J., Verhoef, J., Fluit, A. C. & the Sentry Participants Group. (1999). Prevalence of resistance to MLS antibiotics in 20 European university hospitals participating in the European SENTRY surveillance programme. Journal of Antimicrobial Chemotherapy 43, 783–92.[Abstract/Free Full Text]

4 . Bastos, M. C., Mondino, P. J., Azevedo, M. L., Santos, K. R. & Giambiagi-deMarval, M. (1999). Molecular characterization and transfer among Staphylococcus strains of a plasmid conferring high-level resistance to mupirocin. European Journal of Clinical Microbiology and Infectious Diseases 18, 393–8.[ISI][Medline]

5 . Leski, T. A., Gniadkowski, M., Skoczynska, A., Stefaniuk, E., Trzcinski, K. & Hryniewicz, W. (1999). Outbreak of mupirocinresistant staphylococci in a hospital in Warsaw, Poland, due to plasmid transmission and clonal spread of several strains. Journal of Clinical Microbiology 37, 2781–8.[Abstract/Free Full Text]

6 . Udo, E. E., Farook, V. S., Mokadas, E. M., Jacob, L. E. & Sanyal, S. C. (1998–9). Molecular fingerprinting of mupirocin-resistant methicillin-resistant Staphylococcus aureus from a burn unit. International Journal of Infectious Disease 3, 82–7.

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Received 21 May 2001; returned 9 November 2001; accepted 5 December 2001