J. Antimicrob. Chemother.-2002-Yamakawa-455-65.pdff

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Introduction

Topical application of antimicrobial agents is a useful toolfor 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 manymutational resistances. Thirdly, topical applications areless likely than systemic therapy to cause side effects.

At present, there are several kinds of antimicrobialagent used in topical applications, such as β-lactams, quino-lones, aminoglycosides, macrolides, tetracyclines, mupirocinand fusidic acid. For chemotherapy of infection, antibioticresistance 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, despiteadvances in antimicrobial therapy over the last 20 years.1

The incidence of staphylococci and streptococci resistantto macrolides and aminoglycosides, in particular, hasincreased markedly in recent years.2,3 One of the reasonsfor this is that resistance to these agents is easily spreadhorizontally to other bacteria by R plasmids or trans-posons. Recently, the emergence of plasmid-mediatedmupirocin resistance in methicillin-resistant Staphylococ-

cus aureus (MRSA) has also been reported.4–6

Quinolones have a broad spectrum, display potentantibacterial activity and are bactericidal against Gram-positive and Gram-negative bacteria. Moreover, quinolonesshow no cross-resistance to other classes of antimicrobials.

455

Original articles

In vitro and in vivo antibacterial activity of T-3912, a novelnon-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, wascompared 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 theMIC90 for the clinical isolates. The activity of T-3912 was not influenced by grlA mutation inS. aureus , and the degree of MIC increase of T-3912 for grlA–gyrA double and triple mutantswas lowest among the quinolones tested (nadifloxacin, levofloxacin and ofloxacin). Theinhibitory activity of T-3912 was compared with other quinolones for DNA gyrase andtopoisomerase IV of S. aureus SA113. T-3912 showed the greatest inhibitory activity for bothenzymes among the quinolones tested. The isolation frequency of spontaneous mutantsresistant to T-3912 was < 1.7 109 and < 2.0 109 for S. aureus SA113 and P. acnes JCM6425, respectively. Furthermore, resistance to T-3912 could not be clearly detected in the28th transfer by the serial passage method. T-3912 exhibited more potent bactericidalactivity against S. aureus and P. acnes than nadifloxacin and clindamycin in a short timeperiod. T-3912 in a 1% gel formulation showed good therapeutic activity against a burninfection model caused by S. aureus SA113, P. acnes JCM6425 and multidrug-resistantS. aureus F-2161. These results indicate that T-3912 is potentially a useful quinolone for thetreatment of skin and soft-tissue infections and that its potent bactericidal activity might beable to shorten the treatment period.

*Corresponding author. Tel:81-76-431-8270; Fax:81-76-431-8208; E-mail: [email protected]

© 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 hasproved problematic during and after quinolone therapyfor several types of infection. However, it has been foundthat a new class of des-F(6)-quinolones/non-fluorinatedquinolones has good antibacterial activity.7,8 In attemptingto select a new topical quinolone with a high level ofactivity 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 apotential topical quinolone candidate for development.

In this study, the in vitro and in vivo antibacterial activityof 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 andgentamicin. Ofloxacin (Sigma Chemical, St Louis, MO),clindamycin (Sigma), erythromycin (Sigma) and genta-micin (Schering-Plough, Osaka, Japan) were commerciallyavailable. Nadifloxacin and levofloxacin were prepared inour laboratories. The purity of these quinolones was above99%, as measured by high-performance liquid chromato-graphy.

For in vivo evaluation, the following were used: T-39121% gel, prepared in our laboratory, and commerciallyavailable nadifloxacin 1% cream (Acuatim cream; OtsukaPharmaceutical, 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 collectedfrom various hospitals and research institutes in Japanfrom 1997 to 1998. They included 48 strains of S. aureus,including 23 strains of ofloxacin-resistant MRSA; 53 strainsof 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 thisstudy 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 andF-2161 were obtained from clinical isolates from varioushospitals in Japan.

The nucleotide sequences of gyrA and grlA in thequinolone resistance-determining region of these strainswere determined by dideoxy chain termination methods asreported previously.10

Animals

Three-and-a-half-week-old male ICR strain mice, pur-

chased from Japan SLC (Shizuoka, Japan), were assignedto the study after an acclimatization period of 3 days.

Antibacterial activity

MICs were determined by the standard agar dilutionmethod or the broth microdilution method recommendedby 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% defibrinatedsheep 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 foranaerobic bacteria.12 Almost all strains were tested at afinal inoculum of 104 cfu/spot by using a multipoint inocula-tor (Sakuma Seisakusho, Tokyo, Japan). Aerobic bacteriawere incubated at 37C 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. TheMIC was defined as the lowest antibiotic concentration thatprevented the visible growth of bacteria.

For the broth microdilution method, Mueller–Hinton

broth (MHB, Difco) cation-adjusted with calcium andmagnesium was used. Two-fold dilutions of antibiotics anda final bacterial concentration of c. 5 104 cfu were placedin each well and the plates incubated at 37C 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 SA113were cloned and expressed in Escherichia coli DH5, as afusion protein with maltose binding protein according tothe method described by Tanaka et al.13

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Figure 1. Chemical structure of T-3912.



In vitro and in vivo activity of T-3912

For inhibition of topoisomerase IV, the IC50 was deter-mined as the drug concentration that reduced decatenationactivity by 50%, as seen with drug-free controls. For inhibi-tion of DNA gyrase, the IC50 was determined as the drugconcentration that reduced supercoiling activity, i.e. the

conversion of relaxed pBR322 DNA to the supercoiledform by 50%, as seen with the drug-free controls.

Determination of mutant frequency

The frequencies of occurrence of spontaneous mutantsresistant to T-3912, nadifloxacin, levofloxacin, clindamycin,erythromycin and gentamicin in S. aureus SA113 andP. acnes JCM6425 were determined by spreading a 0.1 mLsample of a culture of each test organism on to MHA platesfor S. aureus and modified GAM agar plates for P. acnes

containing drugs at concentrations of 4 MIC. Afterincubation at 37C for 48 h, the colonies were counted

and the frequency of occurrence of spontaneously resistantmutants 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 abroth microdilution method by exposing bacteria to step-wise increasing concentrations of antibiotics by slightmodification of the method of Entenza et al.15 Selectionof 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 wellscontaining two-fold serial dilutions of each test drug wasinoculated with a final concentration of 105 cfu/mL andthen incubated for 24 h for S. aureus SA113 and for 48 hfor P. acnes JCM6425. In the next step, the well with thehighest antibiotic concentration still showing turbidity wasused to inoculate a new series of microtitre tray. The pro-cedure was repeated, and the MICs were determined for upto 28 passages.

Bactericidal activity

In order to assess bactericidal activity, the log10 reduction

of bacterial counts in a definite time was investigated with arange of drug concentrations of 1–32 MIC, to give aninoculum size of c. 106 cfu/mL at 37C. The incubationperiod was 2 h for S. aureus SA113 and 4 h for P. acnes

JCM6425. After incubation, viable counts were made onsolid agar. Experiments were carried out in triplicate, andthe log10 reduction of viable counts was given as the mean standard deviation.

In vivo therapeutic efficacy

This animal study was approved by the Internal EthicsCommittee of Toyama Chemical on Animal Studies andcarried out according to the procedures stipulated by it.

The therapeutic effect of T-3912 was evaluated by the burninfection 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, wereused. Briefly, using a sterilized cotton swab, bacterial cells

grown on MHA plates were suspended in sterilized physio-logical saline at the desired concentration (confirmed byplacing serial 10-fold dilutions on to MHA and incubatingthe resulting plates for 18 h at 37C). The four-week-oldmale ICR strain mice were anaesthetized by intramuscularinjection of a mixture of 6 mg of ketamine (Sankyo Pharma-ceutical, Tokyo, Japan) per kilogram of body weight and1 mg of xylazine (Bayer, Tokyo, Japan) per kilogram.Their dorsal hair was removed by an electric shaver. Ametal weight (20 mm in diameter) heated to 100C waspressed on the dorsal skin for 5 s. After 1 h, 0.2 mL ofbacterial 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 gentamicin0.1% cream) was applied to the lesion. Mice werehumanely killed 24 h after this for S. aureus SA113 andP. acnes JCM6425 and 48 h after this for S. aureus F-2161.After cleaning the surface of the burned skin lesion using70% alcohol, the burned skin lesions were removed. Theywere then homogenized with sterilized physiological salineand the homogenate diluted with sterilized physiologicalsaline, placed on to MHA plates containing 50 mM MgCl2to avoid carrying over the quinolones. The number of colonies was counted after incubation for 24 h at 37C. The

results were expressed as the mean standard deviation of log cfu per skin sample. The lower limit of detection was104 cfu/skin sample.

Statistical analysis

The log10 reduction of viable cell counts was analysed bythe Tukey procedure with a cutoff of P 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 andGram-negative bacteria were inhibited (MIC50 and MIC90,respectively). The antibacterial activity of T-3912 based onMIC90 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 16 000-fold greaterthan that of nadifloxacin, ofloxacin, levofloxacin, clinda-mycin, erythromycin and gentamicin. The activity ofT-3912 against penicillin-susceptible S. pneumoniae

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T. Yamakawa et al.

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

Organism Antibacterial Range of MIC MIC50 MIC90

(number of strains) agent (mg/L) (mg/L) (mg/L)

S. aureus T-3912 0.00313–0.00625 0.00625 0.00625methicillin-susceptible (25) nadifloxacin 0.0125–0.05 0.025 0.05

ofloxacin 0.2–1.56 0.39 0.78levofloxacin 0.1–0.39 0.2 0.39clindamycin 0.1–>100 0.1 0.2erythromycin 0.2–>100 0.2 >100gentamicin 0.05–100 0.39 100

S. aureus T-3912 0.025–0.2 0.2 0.2methicillin-resistant, nadifloxacin 0.78–1.56 1.56 1.56ofloxacin-resistant (23) ofloxacin 12.5–100 50 50

levofloxacin 3.13–50 25 25clindamycin >100 >100 >100

erythromycin >100 >100 >100gentamicin 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 0.39levofloxacin 0.10–0.39 0.20 0.20clindamycin 0.05–>100 0.1 1.56erythromycin 0.025–>100 0.1 >100gentamicin 0.025–100 0.1 50

S. epidermidis T-3912 0.05–0.2 0.1 0.2ofloxacin-resistant nadifloxacin 0.78–1.56 0.78 1.56(26) ofloxacin 6.25–50 6.25 25

levofloxacin 3.13–25 3.13 12.5clindamycin 0.025–>100 0.1 >100erythromycin 0.05–>100 50 >100gentamicin 0.05–>100 50 100

S. pneumoniae T-3912 0.0125–0.1 0.05 0.1penicillin-susceptible (20) nadifloxacin 0.39–3.13 0.78 1.56

ofloxacin 1.56–6.25 1.56 3.13levofloxacin 0.78–3.13 0.78 1.56clindamycin 0.00313–0.05 0.025 0.025erythromycin 0.00313–3.13 0.025 0.39gentamicin 3.13–12.5 6.25 12.5

S. pneumoniae T-3912 0.025–0.05 0.05 0.05penicillin-resistant (22) nadifloxacin 0.78–1.56 0.78 1.56ofloxacin 0.78–1.56 0.78 1.56levofloxacin 0.39–0.78 0.39 0.39clindamycin 0.025–>100 0.025 >100erythromycin 0.0125–>100 0.05 >100gentamicin 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





was inferior to that of clindamycin and four- to 128-foldgreater than that of nadifloxacin, ofloxacin, levofloxacin,erythromycin and gentamicin. The activity of T-3912against S. pyogenes was comparable to that of erythromycin

and two- to 256-fold greater than that of nadifloxacin,ofloxacin, levofloxacin, clindamycin and gentamicin. Theactivity 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 andreference quinolones against S. aureus having mutations atthe 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- to1024-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– gyrA double mutants and 1024-foldfor the grlA– gyrA triple mutant. In a similar manner, theMIC of T-3912 for the grlA mutant was not different fromthat for the wild-type strain but increased two- to eight-foldfor the grlA– gyrA double mutants and 32-fold for the grlA– gyrA triple mutant. The activity of T-3912 was notinfluenced by the grlA mutation, and the degree ofMIC increase for the grlA– gyrA double mutants and the grlA– gyrA triple mutant in T-3912 was the lowest amongthe quinolones tested.

Table 3 shows the MICs and the inhibitory activity ofT-3912 and other reference quinolones for DNA gyraseand topoisomerase IV obtained from S. aureus SA113. The

IC50 values of T-3912 for DNA gyrase and topoisomeraseIV were 4.50 and 0.617 mg/L, respectively. For both DNAgyrase and topoisomerase IV, T-3912 showed the greatestinhibitory activity among the quinolones tested.

Development of resistance

Table 4 shows the isolation frequency of spontaneousmutants in S. aureus SA113 and P. acnes JCM6425 resistantto T-3912 and other agents.

In S. aureus, the isolation frequency of mutants resistantto T-3912 was 1.7 109, lower than that of gentamicinand levofloxacin and comparable to that of nadifloxacin,clindamycin and erythromycin. In P. acnes, the isolationfrequency of mutants resistant to T-3912 was2.0 109,comparable to that of other agents. In general, the isolationrate of mutants resistant to T-3912 was quite low, similar tothat 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 wastwo-fold for T-3912, four-fold for nadifloxacin, four- to64-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 log10 reduction in bacterial counts ofS. aureus SA113 and P. acnes JCM6425 when exposed toeach agent at concentrations of 1–32MIC.

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Table 1. (Continued)

Organism Antibacterial Range of MIC MIC50 MIC90

(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 3.13levofloxacin 0.1–>100 0.39 3.13clindamycin >100 >100 >100erythromycin 25–>100 >100 >100gentamicin 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


Inoculum size: 104 cfu/spot.



T. Yamakawa et al.

Against S. aureus SA113, the bactericidal activity ofT-3912 was inferior to that of gentamicin but superior tothat of nadifloxacin and clindamycin at concentrationsof 4–32 and 1–32MIC, respectively.

Against P. acnes JCM6425, although the activity ofT-3912 was again inferior to that of gentamicin at a con-centration of 32 MIC, it was superior to that of nadi-floxacin and clindamycin at concentrations of 4–32 and

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Table 2. Antibacterial activity of T-3912 and other quinolones against quinolone-resistant S. aureus

Mutations at QRDRsa MIC (mg/L)b

Strain GyrA GrlA T-3912 nadifloxacin ofloxacin levofloxacin

SA113 (wild-type) 0.0078 0.0313 0.25 0.125CR-3 Ser83

Phe 0.0078 0.0313 1 0.5F-1659 Glu88

Gly Ser80Tyr 0.0156 0.25 4 2

CRCP-9 Glu88Lys Ser80

Phe 0.0625 0.5 8 4F-1614 Ser84

Leu Glu84Lys 0.0625 2 32 16

F-2161 Ser84Leu, Glu88

Lys Ser80Phe 0.25 32 >128 >128

aQuinolone resistance-determining regions.b104 cfu/well.

Table 3. Inhibitory activity of T-3912, nadifloxacin, ofloxacin and levofloxacin

against DNA gyrase and topoisomerase IV obtained from S. aureus SA113

IC50 (mg/L)

Drugs MIC (mg/L)a DNA gyrase topoisomerase IV

T-3912 0.0078 4.50 0.617Nadifloxacin 0.0313 8.83 3.22Ofloxacin 0.25 119 3.59Levofloxacin 0.125 39.7 1.70

a104 cfu/well.

Table 4. Frequency of spontaneous mutants resistant to T-3912 and other agents

Organism Drug MIC (mg/L)a Frequency (109 )(4MIC)

S. aureus SA113 T-3912 0.00625 <1.7nadifloxacin 0.025 <1.7levofloxacin 0.1 420clindamycin 0.1 <1.7erythromycin 0.2 <1.7gentamicin 0.2 1800

P. acnes JCM6425 T-3912 0.05 <2.0nadifloxacin 0.39 <2.0levofloxacin 0.78 <2.0clindamycin 0.1 <2.0erythromycin 0.05 <2.0gentamicin 3.13 <2.0

a104 cfu/spot.




1–32 MIC, respectively. In most cases, gentamicindecreased the viable counts most potently against thesestrains, followed by T-3912 and nadifloxacin. Clindamycinshowed bacteriostatic activity against these strains. T-3912exhibited 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 formulationand each commercially available formulation of the otheragents on an experimental burn infection model caused byS. aureus SA113 and P. acnes JCM6425. For the infectioncaused by S. aureus SA113, T-3912 1% gel, nadifloxacin 1%cream, clindamycin 1% gel and erythromycin 2% gel allsignificantly decreased the bacterial count in the skin lesioncompared with the control (P 0.01). Clindamycin 1% gel

significantly decreased bacterial counts compared withnadifloxacin 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 comparedwith gentamicin 0.1% gel (P 0.01). For the infection

caused by P. acnes JCM6425, once again all drugs testedsignificantly decreased bacterial counts compared with thecontrol (P 0.01), but in this case T-3912 1% gel showedsuperior efficacy in reducing the bacterial count comparedwith nadifloxacin 1% cream, clindamycin 1% gel, erythro-

mycin 2% gel and gentamicin 0.1% gel (P 0.01).Figure 5 shows the case of infection caused by the

grlA– gyrA triple mutant S. aureus F-2161. As shown inthe Figure, only T-3912 1% gel decreased log cfu/skin atthe burned lesion significantly compared with not only thecontrol 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. Mostskin and soft-tissue infections are caused by Gram-positive

organisms, i.e. S. aureus and the β-haemolytic Streptococcusspecies. MRSA, in particular, remains a serious cause of infection. Therefore, we evaluated the antibacterial activ-ity of T-3912 against Gram-positive organisms, includingdrug-resistant bacteria.

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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 serialpassage method: , T-3912;, nadifloxacin;, levofloxacin;, clindamycin;, erythromycin;, gentamicin.



T. Yamakawa et al.

T-3912 showed the greatest antibacterial activity againstclinical isolates of Gram-positive organisms, i.e. methicillin-susceptible S. aureus, ofloxacin-resistant MRSA, S. epider-

midis, ofloxacin-resistant S. epidermidis, penicillin-resistantS. pneumoniae and P. acnes. In addition, T-3912 showedimproved activity against S. aureus with mutations in theDNA gyrase and topoisomerase IV.

The target molecules of quinolones are DNA gyraseand topoisomerase IV. The development of quinoloneresistance is caused by point mutations in discrete regionsof the DNA gyrase and topoisomerase IV genes called the

quinolone resistance-determining regions.17,18 Quinoloneresistance in S. aureus arises through mutation of the parC

( grlA) or parE ( grlB) genes before changes in the DNAgyrase genes take place, indicating that topoisomerase IVis the primary target and that DNA gyrase is the secondarytarget 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 alterfor the grlA mutant compared with the wild type, and thedegree of MIC increase for the grlA– gyrA double mutantand 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 S.D. (n 3); P 0.05. (a) Dotted line represents the control after incubation for 2 h inS. aureus SA113;, T-3912 (MIC 0.0078 mg/L); , nadifloxacin (MIC 0.0625 mg/L); , clindamycin (MIC 0.125 mg/L); , gentamicin(MIC 0.5 mg/L); T-3912 versus nadifloxacin (4–32 MIC), T-3912 versus clindamycin (1–32 MIC), T-3912 versus gentamicin(1–32 MIC), nadifloxacin versus clindamycin (1–32 MIC), nadifloxacin versus gentamicin (1–32 MIC), clindamycin versusgentamicin (1–32 MIC). (b) Dotted line represents the control after incubation for 4 h in P. acnes JCM6425; , T-3912 (MIC0.0313 mg/L); , nadifloxacin (MIC 1 mg/L); , clindamycin (MIC 0.0625 mg/L); , gentamicin (MIC 4 mg/L); T-3912 versusnadifloxacin (4–32 MIC), T-3912 versus clindamycin (2–32 MIC), T-3912 versus gentamicin (32 MIC), nadifloxacin versusgentamicin (8–32MIC), clindamycin versus gentamicin (4–32 MIC).




quinolones tested. Secondly, the IC50 of T-3912 for bothtopoisomerase IV and DNA gyrase was lowest among thequinolones tested, reflecting the MIC. Thirdly, the isolationfrequency of spontaneous mutants resistant to T-3912 wasquite low, and resistance to T-3912 was not clearly detected

using the serial passage method. These results indicate thatthe target of T-3912 may be both DNA gyrase and topoiso-merase IV in S. aureus. Although further investigation isneeded on this matter, from the viewpoint of bacterialresistance, this characteristic is very advantageous for thetreatment of infectious diseases, including skin and soft-tissue infections.

In skin and soft-tissue infections, it is also necessary toreduce the bacterial counts as rapidly as possible from theinfectious lesion. Therefore, rapid bactericidal activity isone of the important characteristics of topical agentsrequired for an optimal in vivo therapeutic effect. T-3912

showed greater bactericidal activity than nadifloxacin andclindamycin against S. aureus and P. acnes, which are thetypical pathogens of skin and soft-tissue infections andacne, in a short time period. In addition, T-3912 1% gelshowed a superior therapeutic effect in the burn infectionmodel 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 MRSAhas increased, with strains shown to cause up to 21% of skininfection.26 Therefore, efficacy against MRSA will becomemore important for the treatment of skin infections. In

the burn infection model of S. aureus F-2161, a highlymultidrug-resistant strain including methicillin resistancewith grlA and gyrA triple mutation, only T-3912 1% gelshowed a therapeutic effect, which was reflected in itsMIC.

On the other hand, it is reasonable to consider that theimportant factors determining the in vivo efficacy are notonly antibacterial activity and bactericidal activity but alsothe tissue distribution of these drugs depending on formu-lation. Indeed, the bactericidal activity of gentamicin wasmore potent than that of T-3912, but the therapeutic effectof gentamicin was inferior to that of T-3912. This resultindicated that the tissue concentration of gentamicin in the

infectious lesion was lower than that of T-3912. Moreover,the permeability of gentamicin through the stratumcorneum 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 thesetopical agents and their formulations in skin.

In conclusion, T-3912 is a potentially useful quinolonefor the treatment of skin and soft-tissue infections, and itspotent bactericidal activity might be able to shorten thetreatment 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. Kurodaand H. Kawabuchi for the supply of chemical compounds.We are also grateful to S. Kato, M. Katai and M. Kadonofor the preparation of formulations.

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Figure 4. Effect of T-3912 on lesions subcutaneously infectedwith (a) S. aureus SA113 and (b) P. acnes JCM6425 in mice. Micewere infected subcutaneously with 0.2 mL of bacterial suspen-sion (S. aureus SA113, 1.0 107 cfu/mouse; P. acnes JCM6425,5.0 106 cfu/mouse). Drugs were applied at 2 h after infection(10 mg gel or cream per mouse). Viable cells were counted at 24 hafter 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. TheMICs 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 carriedout using the Tukey procedure.




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